Multiple promoter expression cassettes for simultaneous delivery of RNAi agents targeted to Hepatitis C virus

ABSTRACT

The present invention provides multiple-promoter expression cassettes for simultaneous delivery of RNAi, preferably to mammalian cells in vivo.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 60/553,920, filed Mar. 17, 2004, and of U.S. Provisional PatentApplication Ser. No. 60/550,504, filed Mar. 5, 2004, both of which areherein incorporated by reference.

BACKGROUND OF THE INVENTION

Utilization of double-stranded RNA to inhibit gene expression in asequence-specific manner has revolutionized the drug discovery industry.In mammals, RNA interference, or RNAi, is mediated by 19- to29-nucleotide long, double-stranded RNA molecules referred to as smallinterfering RNAs that are derived by enzymatic cleavage of long,double-stranded RNA within cells in vivo. RNAi agents can be synthesizedchemically or enzymatically outside of cells and subsequently deliveredto cells (see, e.g., Fire, et al., Nature, 391:806-11 (1998); Tuschl, etal., Genes and Dev., 13:3191-97 (1999); and Elbashir, et al., Nature,411:494-498 (2001)); or can be expressed in vivo by an appropriatevector in cells (see, e.g., McCaffrey, et al. Nature Biotech. 21(6):639-644 (2003)).

However, in vivo delivery of unmodified RNAi as an effective therapeuticfor use in humans faces a number of technical hurdles. First, due tocellular and serum nucleases, the half life of RNA injected in vivo isonly about 70 seconds (see, e.g., Kurreck, Eur. J. Bioch. 270:1628-44(2003)). Efforts have been made to increase stability of injected RNA bythe use of chemical modifications; however, there are severaloccurrences in which chemical alterations led to increased cytotoxiceffects. In one specific example, cells were intolerant to doses of anRNAi duplex in which every second phosphate was replaced byphosphorothioate (Harborth, et al., Antisense Nucleic Acid Drug Rev.13(2): 83-105 (2003)). Other hurdles include providing tissue-specificdelivery, as well as being able to deliver the RNAi agents in amountssufficient to elicit a therapeutic response, but that are not toxic.

Several options are being explored for RNAi delivery, including the useof viral-based vector systems that can infect target cells, and deliverand express RNAi molecules in situ. Typically, small RNAs ofapproximately 70 nucleotides are transcribed as short hairpin precursors(shRNA) from a viral vector backbone. Once transcribed, the shRNA areprocessed by the enzyme Dicer into the appropriate active RNAi species.Viral-based delivery approaches attempt to exploit the targetingproperties of viruses to generate tissue specificity and onceappropriately targeted, rely upon the endogenous cellular machinery togenerate sufficient levels of the RNAi species to achieve atherapeutically effective dose.

Currently, the most commonly used viruses for delivery of targetsequences are those based upon systems evolved from retrovirus, herpessimplex virus (HSV) or adenovirus (Ad). All of these vectors canaccommodate rather large inserts and can be produced in therapeuticallyrelevant titers. However, in all systems, there are concerns relating todevelopment of cancer (Cavazzana-Calvo, et al., Science, 288:669-72(2000)), as well as undesirable host immune responses and resultingtoxicity in patients. Another virus that is useful for delivering RNAiis adeno-associated virus (AAV).

One useful application of RNAi therapeutics is as an anti-viral agent.In general, RNA viruses depend on RNA/DNA-dependent RNA polymerase forreplication. Such RNA/DNA polymerases replicate the viral genome withcomparatively low fidelity, the functional consequence of which producesgenomes with an exceptionally high number of mutations. This results inthe ability of rapidly evolving progeny virions to evade commonimmunological and chemical antiviral agents. Thus, similar to theeffects observed with small molecule therapeutics, the relative potencyand efficacy of the RNAi therapeutic may decrease as a result of viralevolution during long term treatment. In one study, HIV escape mutantsthat contained a single nucleotide change appeared 35 days afterdelivery of an expressed shRNA (Boden, et al., J. Virol. 77(21):11531-11535 (2003)). In another study, poliovirus escape mutants couldbe detected in as little as 54 hours post-infection in cells that hadbeen transfected with pre-synthesized RNAi. Yet, the simultaneousdelivery of two RNAis against multiple target sequences within the virussignificantly delayed the onset of escape variants (see Gitlin, et al.,Nature. 418: 430-434 (2002)).

Thus, there is a need in the art to develop stable, effective RNAitherapeutics. The present invention satisfies this need in the art.

SUMMARY OF THE INVENTION

The present invention is directed to innovative compositions and methodsfor delivering RNAi species or agents to target cells. The RNAi speciesare part of a multiple promoter expression construct preferablydelivered via a viral delivery system. Because three or more RNAi agentsare used per construct, the present invention is particularly useful inaddressing organisms with target genes that have sequence differences(SNPs) between variants, where each of the introduced RNAi agents cantarget one or more subset of variants. Similarly, the compositions andmethods of the present invention are also useful in treating diseasestates caused by rapidly mutating pathogens, such as diseases caused byRNA-based viral agents; that is, it is less likely that viral escapemutants will be able to avoid the effect of three or more different RNAisequences.

Thus, embodiments of the present invention provide a multiple promoterexpression cassette comprising: at least three promoter/RNAi/terminatorcomponents, where each promoter/RNAi/terminator component comprises apromoter element, a terminator element, and an RNAi species operablylinked to the promoter element and the terminator element, where thesequence of each of the RNAi species is different from one another. Invarious preferred aspects of this embodiment, the sequence of each ofthe promoter elements in the multiple-promoter expression cassette isdifferent from one another. In other aspects of this embodiment, thesequence of each of the terminator elements in the multiple-promoterexpression cassette is different from one another and/or each terminatorelement is taken from the same gene as the promoter element with whichit is paired in nature. In addition, in one aspect of this embodiment,the present invention provides a multiple promoter expression constructthat contains elements necessary for packaging of the therapeutic vectorinto infectious virus particles.

In another embodiment of the present invention, there is provided amethod of treating one or more nucleic acid targets that are expressedin a cell comprising: incorporating a multiple promoter RNAi expressioncassette that expresses three or more RNAi agents to inhibit one or morenucleic acid targets into a viral vector in order to produce a viralRNAi delivery construct; packaging the viral RNAi delivery constructinto viral particles; delivering the viral particles to the cell; andexpressing three or more RNAi agents from the multiple promoterexpression cassette. In various aspects of this embodiment of theinvention, the one or more nucleic acid targets that are expressed aregenes necessary for the initiation or maintenance of a disease state,such as a cancerous state, in the cell. In other aspects of thisembodiment of the invention, the one or more nucleic acid targets thatare expressed are genes necessary for the infection or maintenance ofinfection of the cell by a pathogen. Alternatively, themultiple-promoter RNAi expression cassette may be provided in anon-viral vector and delivered to cells via non-viral methods known inthe art.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features, advantages andobjects of the present invention are attained and can be understood indetail, a more particular description of the invention, brieflysummarized above, may be had by reference to the embodiments that areillustrated in the appended drawings. It is to be noted, however, thatthe appended drawings illustrate only certain embodiments of thisinvention and are therefore not to be considered limiting of its scope,for the present invention may admit to other equally effectiveembodiments.

FIG. 1 is a simplified block diagram of one embodiment of a method fordelivering RNAi species according to the present invention.

FIGS. 2A and 2B are simplified schematic representations of embodimentsof the multiple-promoter RNAi expression cassette of the presentinvention.

FIGS. 3A and 3B show two embodiments of multiple expression cassettesthat deliver RNAi agents as shRNA precursors. FIG. 3C shows anembodiment of a multiple expression cassettes comprising stuffer regionsinserted between promoter/RNAi/terminator components. FIGS. 3D and 3Eshow embodiments of multiple-promoter RNAi expression cassettes thatdeliver RNAi without a shRNA precursor.

FIG. 4A is a simplified representation of one method of producingmultiple-promoter RNAi expression vectors packaged in viral particles.FIG. 4B is a simplified representation of another method of producingmultiple-promoter RNAi expression vectors packaged in viral particles.

FIG. 5 is a schematic of one embodiment of a test recombinant AAV (rAAV)expression construct and a luciferase reporter construct.

FIG. 6 is a schematic of a self-complementary (scAAV) RNAi expressionvector according to one embodiment of the present invention.

FIG. 7 is a schematic of a representative promoter testing construct anda reporter construct.

FIG. 8A is a schematic of the HCV genome showing the position of RNAiagents tested in experiments described herein. 8B is a schematic of aluciferase-HCV fusion replicon containing genetic elements fornon-structural proteins. 8C is a schematic of a luciferase-HCV fusionreplicon containing HCV genetic elements for structural andnon-structural proteins.

FIG. 9 is a schematic of two luciferase-HCV fusion reporter plasmidsuseful for testing RNAi agents. The construct on the left comprises one100 bp HCV sequence fused to a luciferase gene; while the construct onthe right comprises 3 different 100 bp HCV sequences fused to aluciferase gene. RNAi agents targeting a sequence contained within the100 bp region will, if effective, degrade the HCV-luciferasetranscription product, thus decreasing (perhaps eliminating) luciferaseexpression.

FIG. 10 is a graphic illustration of one embodiment of a triple promotercassette showing unique restriction sites useful for modular assembly ofvarious RNAi agents, promoter elements and terminator elements.

FIG. 11A is an example of a sequence (SEQ ID NO 31) of the triplepromoter cassette type shown in FIG. 3B. FIGS. 11B/11C is an example ofa sequence (SEQ ID NO 32) of the triple promoter cassette type shown inFIG. 3C.

FIG. 12 shows the results of inhibition of luciferase expressionmeasured in relative light units (RLU) by different RNAi agentstargeting five different 100 bp regions of HCV.

FIG. 13 shows the results of inhibition of luciferase expression bydifferent RNAi agents expressed as a percent inhibition value.

FIG. 14 shows the reproducibility of the results of experimentsperformed testing four different RNAi agents targeting various segmentsof a 100 bp sequence from the 5′ region of HCV.

FIG. 15 shows the change in percent inhibition of luciferase expression24 hours post transfection and 48 hours post transfection for fivedifferent RNAi agents targeting various segments of a 100 bp sequence inthe 5′ region of HCV.

FIG. 16 shows the change in percent inhibition of luciferase expression44 hours post transfection and 72 hours post transfection for twodifferent RNAi agents targeting various segments of a 100 bp sequence inthe 5′ region of HCV, five different RNAi agents targeting varioussegments of a 100 bp sequence in the 3′ region of HCV, and one RNAiagent targeting a segment of a 100 bp sequence in the open reading frameregion of HCV.

FIGS. 17A and 17B show data assessing the strength of three Pol IIIpromoters. An shRNA sequence specific to firefly luciferase mRNA(McCfrey at al., 2002) was inserted under control of indicated Pol IIIpromoter. Resulting plasmid DNA was co-transfected together with aluciferase reporter plasmid either in Huh7 cells (FIG. 17A) or 293 cells(FIG. 17B). Luciferase levels were measured 72 hrs post transfection. Intriple-promoter constructs from FIG. 3B (right three constructs on eachpanel) the promoter driving shRNA is indicated.

FIG. 18 shows the inhibition of luciferase expression with differentsiRNA agents in luciferase-HCV fusion reporter plasmid assay. Theluciferase-HCV reporter plasmid was co-transfected with each siRNA agentinto Huh7 cells, and luciferase activity was measured 48 hrs later.

FIG. 19 shows the activity of selected siRNA agents against a subgenomicLuciferase-HCV fusion replicon. Tested siRNA agents were transfectedinto 29Σ cells together with a trace amount of pGL3Control DNA (as acontrol for transfection efficiency). Both renilla and fireflyluciferase levels were measured 48 hours later.

FIG. 20 shows the percent inhibition of luciferase signal from aluciferase-HCV reporter plasmid after co-transfection with plasmidscomprising promoter/shRNA cassettes containing one or two activepromoters.

FIG. 21 shows the percent inhibition of luciferase signal from aluciferase-HCV reporter plasmid containing coding region C12 (top),coding region C-9 (middle), or 5′6 region (bottom) after co-transfectionwith plasmids comprising one, two or three active promoter/shRNAcassettes.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmethodology, products, apparatus and factors described, as such methods,apparatus and formulations may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention which will be limited only by appendedclaims.

As used herein, the singular forms “a,” “and,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a factor” refers to one or mixtures of factors,and reference to “the method of production” includes reference toequivalent steps and methods known to those skilled in the art, and soforth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference, without limitation, for thepurpose of describing and disclosing devices, formulations andmethodologies which are described in the publication and which might beused in connection with the presently described invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

The present invention is directed to innovative, robust geneticcompositions and methods to deliver at least three different RNAi agentssimultaneously to a cell using a single expression construct. Thecompositions and methods provide stable, lasting inhibition of targetnucleic acids.

Generally, conventional methods of molecular biology, microbiology,recombinant DNA techniques, cell biology, and virology within the skillof the art are employed in the present invention. Such techniques areexplained fully in the literature, see, e.g., Maniatis, Fritsch &Sambrook, Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: APractical Approach, Volumes I and II (D. N. Glover, ed. 1985);Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Nucleic AcidHybridization (B. D. Hames & S. J. Higgins, eds. (1984)); Animal CellCulture (R. I. Freshney, ed. 1986); and RNA Viruses: A practicalApproach, (Alan, J. Cann, Ed., Oxford University Press, 2000).

A “vector” is a replicon, such as plasmid, phage, viral construct orcosmid, to which another DNA segment may be attached. Vectors are usedto transduce and express the DNA segment in cells.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase in a cell and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNAs, small nuclear of nucleolar RNAs or any kind of RNAtranscribed by any class of any RNA polymerase I, II or III.

A cell has been “transformed”, “transduced” or “transfected” by anexogenous or heterologous nucleic acid or vector when such nucleic acidhas been introduced inside the cell, for example, as a complex withtransfection reagents or packaged in viral particles. The transformingDNA may or may not be integrated (covalently linked) into the genome ofthe cell. With respect to eukaryotic cells, a stably transformed cell isone in which the transforming DNA has become integrated into a host cellchromosome or is maintained extra-chromosomally so that the transformingDNA is inherited by daughter cells during cell replication or is anon-replicating, differentiated cell in which a persistent episome ispresent.

The term “RNA interference” or “RNAi” refers generally to a process inwhich a double-stranded RNA molecule or a short hairpin RNA changes theexpression of a nucleic acid sequence with which they share substantialor total homology. The term “RNA species” or “RNAi agent” refers to adistinct RNA sequence that elicits RNAi; and the term “RNAi expressioncassette” refers to a cassette according to embodiments of the presentinvention comprising three or more RNAi species.

FIG. 1 is a simplified flow chart showing the steps of one method 100 inwhich the multiple-promoter RNAi expression constructs according to thepresent invention may be used. First, in step 200, a multiple-promoterRNAi expression cassette targeting a particular disease target isconstructed. Next, in step 300, the multiple-promoter RNAi expressioncassette is ligated into an appropriate viral delivery construct. Theviral RNAi expression delivery construct is then packaged into viralparticles at step 400, and the viral particles are delivered to thetarget cells to be treated at step 500. Details for each of these stepsand the components involved are presented infra.

The viral-based multiple-promoter RNAi expression constructs accordingto the present invention can be generated synthetically or enzymaticallyby a number of different protocols known to those of skill in the artand purified using standard recombinant DNA techniques as described in,for example, Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), andunder regulations described in, e.g., United States Dept. of HHS,National Institute of Health (NIH) Guidelines for Recombinant DNAResearch. In a preferred embodiment, the multiple-promoter RNAiexpression cassettes are synthesized using phosphoramidite, or analogouschemistry using protocols well known in the art.

FIGS. 2A and 2B are simplified schematics of multiple-promoter RNAiexpression cassettes according to embodiments of the present invention.FIG. 2A shows an embodiment of a multiple-promoter expression cassette(10) with three promoter/RNAi/terminator components (shown at 20), andFIG. 2B shows an embodiment of a multiple-promoter expression cassette(10) with five promoter/RNAi/terminator components (shown at 20). P1,P2, P3, P4 and P5 represent promoter elements. RNAi1, RNAi2, RNAi3,RNAi4 and RNAi5 represent sequences for five different RNAi species. T1,T2, T3, T4, and T5 represent termination elements. The multiple-promoterRNAi expression cassettes according to the present invention may containthree or more promoter/RNAi/terminator components where the number ofpromoter/RNAi/terminator components included in any multiple-promoterRNAi expression cassette is limited by, e.g., packaging size of thedelivery system chosen (for example, some viruses, such as AAV, haverelatively strict size limitations); cell toxicity, and maximumeffectiveness (i.e. when, for example, expression of four RNAi sequencesis as effective therapeutically as the expression of ten RNAisequences).

The three or more RNAi species in the promoter/RNAi/terminatorcomponents comprising a cassette all have different sequences; that isRNAi1, RNAi2, RNAi3, RNAi4 and RNAi5 are all different from one another.However, the promoter elements in any cassette may be the same (that is,e.g., the sequence of two or more of P1, P2, P3, P4 and P5 may be thesame); all the promoters within any cassette may be different from oneanother; or there may be a combination of promoter elements representedonly once and promoter elements represented two times or more within anycassette. Similarly, the termination elements in any cassette may be thesame (that is, e.g., the sequence of two or more of T1, T2, T3, T4 andT5 may be the same, such as contiguous stretches of 4 or more Tresidues); all the termination elements within any cassette may bedifferent from one another; or there may be a combination of terminationelements represented only once and termination elements represented twotimes or more within any cassette. Preferably, the promoter elements andtermination elements in each promoter/RNAi/terminator componentcomprising any cassette are all different to decrease the likelihood ofDNA recombination events between components and/or cassettes. Further,in a preferred embodiment, the promoter element and termination elementused in each promoter/RNAi/terminator component are matched to eachother; that is, the promoter and terminator elements are taken from thesame gene in which they occur naturally.

FIGS. 3A, 3B and 3C show multiple-promoter RNAi expression constructscomprising alternative embodiments of multiple-promoter RNAi expressioncassettes that express short shRNAs. shRNAs are short duplexes where thesense and antisense strands are linked by a hairpin loop. Onceexpressed, shRNAs are processed into RNAi species. Boxes A, B and Crepresent three different promoter elements, and the arrows indicate thedirection of transcription. TERM 1, TERM 2, and TERM 3 represent threedifferent termination sequences, and shRNA-1, shRNA-2 and shRNA-3represent three different shRNA species. The multiple-promoter RNAiexpression cassettes in the embodiments extend from the box marked A tothe arrow marked Term3. FIG. 3A shows each of the threepromoter/RNAi/terminator components (20) in the same orientation withinthe cassette, while FIG. 3B shows the promoter/RNAi/terminatorcomponents for shRNA-1 and shRNA-2 in one orientation, and thepromoter/RNAi/terminator component for shRNA-3 in the oppositeorientation (i.e., transcription takes place on both strands of thecassette).

FIG. 3C shows each of the cassettes separated by a region of DNA toincrease the distance between promoter/RNAi/terminator components. Theinserted DNA, known as “stuffer” DNA, can be any length between 5-5000nucleotides. There can be one or more stuffer fragments betweenpromoters. In the case of multiple stuffer fragments, they can be thesame or different lengths. The stuffer DNA fragments are preferablydifferent sequences. The stuffer DNA fragments may be used to increasethe size of the multiple promoter cassette of the present invention inorder to allow it to fit appropriately into a corresponding deliveryvector. The length of the stuffer is dictated by the size requirementsof the particular vector associated with the multiple promoter cassette.For example, in one embodiment the stuffer fragments total 4000nucleotides (nt) in order to appropriately fulfill the size requirementsof the AAV vector. In another embodiment, the stuffer fragments total2000 nt in order to appropriately fulfill the size requirements of theself complementary AAV vector. Other variations may be used as well.

FIGS. 3D and 3E show multiple-promoter RNAi expression constructscomprising alternative embodiments of multiple-promoter RNAi expressioncassettes that express RNAi species without a hairpin loop. In bothfigures, P1, P2, P3, P4, P5 and P6 represent promoter elements (witharrows indicating the direction of transcription); and T1, T2, T3, T4,T5, and T6 represent termination elements. Also in both figures, RNAi1sense and RNAi1 antisense (a/s) are complements, RNAi2 sense and RNAi2a/s are complements, and RNAi3 sense and RNAi3 a/s are complements.

In the embodiment shown in FIG. 3D, all three RNAi sense sequences aretranscribed from one strand (via P1, P2 and P3), while the three RNAia/s sequences are transcribed from the complementary strand (via P4, P5,P6). In this particular embodiment, the termination element of RNAi1 a/s(T4) falls between promoter P1 and the RNAi 1 sense sequence; while thetermination element of RNAi1 sense (T1) falls between the RNAi 1 a/ssequence and its promoter, P4. This motif is repeated such that if thetop strand shown in FIG. 3D is designated the (+) strand and the bottomstrand is designated the (−) strand, the elements encountered movingfrom left to right would be P1(+), T4(−), RNAi1 (sense and a/s), T1(+),P4(−), P2(+), T5(−), RNAi2 (sense and a/s), T2(+), P5(−), P3(+), T6(−),RNAi3 (sense and a/s), T3(+), and P6(−).

In an alternative embodiment shown in FIG. 3E, all RNAi sense andantisense sequences are transcribed from the same strand. One skilled inthe art appreciates that any of the embodiments of the multiple-promoterRNAi expression cassettes shown in FIGS. 3A through 3E may be used forcertain applications, as well as combinations or variations thereof.

In some embodiments, promoters of variable strength may be employed. Forexample, use of three or more strong promoters (such as a Pol III-typepromoter) may tax the cell, by, e.g., depleting the pool of availablenucleotides or other cellular components needed for transcription. Inaddition or alternatively, use of several strong promoters may cause atoxic level of expression of RNAi agents in the cell. Thus, in someembodiments one or more of the promoters in the multiple-promoter RNAiexpression cassette may be weaker than other promoters in the cassette,or all promoters in the cassette may express RNAi agents at less than amaximum rate. Promoters also may or may not be modified using moleculartechniques, or otherwise, e.g., through regulation elements, to attainweaker levels of transcription.

Promoters may be tissue-specific or cell-specific. The term “tissuespecific” as it applies to a promoter refers to a promoter that iscapable of directing selective expression of a nucleotide sequence ofinterest to a specific type of tissue (e.g., liver) in the relativeabsence of expression of the same nucleotide sequence of interest in adifferent type of tissue (e.g., brain). Such tissue specific promotersinclude promoters such as Ick, myogenin, or thy1. The term“cell-specific” as applied to a promoter refers to a promoter which iscapable of directing selective expression of a nucleotide sequence ofinterest in a specific type of cell in the relative absence ofexpression of the same nucleotide sequence of interest in a differenttype of cell within the same tissue (see, e.g., Higashibata, et al., J.Bone Miner. Res. January 19(1):78-88 (2004); Hoggatt, et al., Circ.Res., December 91(12):1151-59 (2002); Sohal, et al., Circ. Res. July89(1):20-25 (2001); and Zhang, et al., Genome Res. January 14(1):79-89(2004)). The term “cell-specific” when applied to a promoter also meansa promoter capable of promoting selective expression of a nucleotidesequence of interest in a region within a single tissue. Alternatively,promoters may be constitutive or regulatable. Additionally, promotersmay be modified so as to possess different specificities.

The term “constitutive” when made in reference to a promoter means thatthe promoter is capable of directing transcription of an operably linkednucleic acid sequence in the absence of a stimulus (e.g., heat shock,chemicals, light, etc.). Typically, constitutive promoters are capableof directing expression of a coding sequence in substantially any celland any tissue. The promoters used to transcribe the RNAi speciespreferably are constitutive promoters, such as the promoters forubiquitin, CMV, β-actin, histone H4, EF-1alfa or pgk genes controlled byRNA polymerase II, or promoter elements controlled by RNA polymerase I.In preferred embodiments, promoter elements controlled by RNA polymeraseIII are used, such as the U6 promoters (U6-1, U6-8, U6-9, e.g.), H1promoter, 7SL promoter, the human Y promoters (hY1, hY3, hY4 (seeMaraia, et al., Nucleic Acids Res 22(15):3045-52 (1994)) and hY5 (seeMaraia, et al., Nucleic Acids Res 24(18):3552-59 (1994)), the humanMRP-7-2 promoter, Adenovirus VA1 promoter, human tRNA promoters, the 5sribosomal RNA promoters, as well as functional hybrids and combinationsof any of these promoters.

Alternatively in some embodiments it may be optimal to select promotersthat allow for inducible expression of the RNAi species. A number ofsystems for the inducible expression using such promoters are known inthe art, including but not limited to the tetracycline responsive systemand the lac operator-repressor system (see WO 03/022052 A1; and US2002/0162126 A1), the ecdysone regulated system, or promoters regulatedby glucocorticoids, progestins, estrogen, RU-486, steroids, thyroidhormones, cyclic AMP, cytokines, the calciferol family of regulators, orthe metallothionein promoter (regulated by inorganic metals).

One or more enhancers also may be present in the viral multiple-promoterRNAi expression construct to increase expression of the gene ofinterest. Enhancers appropriate for use in embodiments of the presentinvention include the Apo E HCR enhancer, the CMV enhancer that has beendescribed recently (see, Xia et al, Nucleic Acids Res 31-17 (2003)), andother enhancers known to those skilled in the art.

In one embodiment this invention, ApoE enhancer elements may be added tothe multiple promoter cassette of the present invention. The ApoEenhancer is an enhancer element of approximately 155 base pairs (bp)derived from apolipoprotein E or ApoE. One or more copies of the ApoEenhancer may be added upstream or downstream of the first, second and/orthird promoters (or upstream or downstream of more than three promoters,if present) in the multiple promoter cassette of this invention. ApoE isan apolipoprotein that mediates binding, internalization and catabolismof lipoprotein particles and is a ligand for the low-density lipoprotein(ApoB/E) receptor and for the ApoE receptor of hepatic tissues. Thegenetic enhancer associated with the ApoE gene is a eukaryotic controlelement that can increase transcription of a nucleic acid specificallyin the liver. The ApoE enhancer may be located up to 2000 nucleotidesupstream or downstream of a liver-specific promoter, and may be presentin one or more copies.

The RNAi sequences encoded by the multiple-promoter RNAi expressioncassettes of the present invention result in the expression of smallinterfering RNAs that are short, double-stranded RNAs that are not toxicin mammalian cells. There is no particular limitation in the length ofthe RNAi species of the present invention as long as they do not showcellular toxicity. RNAis can be, for example, 15 to 49 bp in length,preferably 15 to 35 bp in length, and are more preferably 19 to 29 bp inlength. The double-stranded RNA portions of RNAis may be completelyhomologous, or may contain non-paired portions due to sequence mismatch(the corresponding nucleotides on each strand are not complementary),bulge (lack of a corresponding complementary nucleotide on one strand),and the like. Such non-paired portions can be tolerated to the extentthat they do not significantly interfere with RNAi duplex formation orefficacy.

The termini of an RNAi species according to the present invention may beblunt or cohesive (overhanging) as long as the RNAi effectively silencesthe target gene. The cohesive (overhanging) end structure is not limitedonly to a 3′ overhang, but a 5′ overhanging structure may be included aslong as the resulting RNAi is capable of inducing the RNAi effect. Inaddition, the number of overhanging nucleotides may be any number aslong as the resulting RNAi is capable of inducing the RNAi effect. Forexample, if present, the overhang may consist of 1 to 8 nucleotides;preferably it consists of 2 to 4 nucleotides.

The RNAi species utilized in the present invention may have a stem-loopstructured precursor (shRNA) in which the ends of the double-strandedRNA are connected by a single-stranded, linker RNA. The length of thesingle-stranded loop portion of the shRNA may be 5 to 20 bp in length,and is preferably 5 to 9 bp in length.

Any transcribed nucleic acid sequence may be a target for themultiple-promoter RNAi expression cassettes of the present invention.Likely targets for the RNAi are genes such as but not limited todevelopmental genes (e.g., adhesion molecules, cyclin kinase inhibitors,Wnt family members, Pax family members, Winged helix family members, Hoxfamily members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2,CBL, CSF1R, ERBA, ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR,HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1,PML, RET, SRC, TAL1, TCL3, and YES); tumor suppressor genes (e.g., APC,BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53, and WT1); and enzymes(e.g., ACC synthases and oxidases, ACP desaturases and hydroxylases,ADP-glucose pyrophorylases, ATPases, alcohol dehydrogenases, amylases,amyloglucosidases, catalases, cellulases, chalcone synthases,chitinases, cyclooxygenases, decarboxylases, dextrinases, DNA and RNApolymerases, galactosidases, glucanases, glucose oxidases, granule-boundstarch synthases, GTPases, helicases, hemicellulases, integrases,inulinases, invertases, isomerases, kinases, lactases, lipases,lipoxygenases, lysozymes, nopaline synthases, octopine synthases,pectinesterases, peroxidases, phosphatases, phospholipases,phosphorylases, phytases, plant growth regulator synthases,polygalacturonases, proteinases and peptidases, pullanases,recombinases, reverse transcriptases, RUBISCOs, topoisomerases, andxylanases); viral structural genes such as capsid and envelope proteins;bacterial genes such as those involved in replication or structuralfeatures, or genes from other pathogens that are involved in replicationor structural features. In addition, the multiple-promoter RNAiexpression cassettes of the present invention may be used to targetspecific sequences that are unique to alleles responsible for pathologyin autosomal dominant diseases such as SCA, the allele responsible forHuntington's Disease, or the collagen gene alleles responsible forosteogenesis imperfecta. An important aspect of the invention is thatviral infections cleared by siRNA can result in no harm to the infectedcells (Gitlin, et. al. Nature 418: 430-434 (2002)). This feature of thepresent invention distinguishes it from prior art methods, whereclearance of virus from the mammalian host causes destruction ofinfected cells, either by the action of the immune system or byapoptosis induced by the virus (Guidotti et. al. Annu. Rev. Immunol. 19:65-91 (2001)). Thus, this aspect of the present invention provides aneffective RNAi agent of noncytopathic viral clearance.

The sequences for the RNAi species are selected based upon the geneticsequence of the target nucleic acid sequence; and preferably are basedon regions of target nucleic acid sequences that are conserved. Forexample, in the case of selecting RNAi sequences for treating a viralinfection or for constructing an RNAi vaccine, the sequences chosenpreferably are those that are conserved between species or evensubspecies of the virus. As viruses are known to mutate rapidly,selection of conserved sequences is likely to preserve the efficacy ofthe RNAi over time. In the case of selection of RNAi sequences to treatcancer or other diseases, the sequences preferably are those that areconserved between variants of genes or oncogenes.

Methods of alignment of sequences for comparison and RNAi sequenceselection are well known in the art. The determination of percentidentity between two or more sequences can be accomplished using amathematical algorithm. Preferred, non-limiting examples of suchmathematical algorithms are the algorithm of Myers and Miller (1988);the search-for-similarity-method of Pearson and Lipman (1988); and thatof Karlin and Altschul (1993). Preferably, computer implementations ofthese mathematical algorithms are utilized. Such implementationsinclude, but are not limited to: CLUSTAL in the PC/Gene program(available from Intelligenetics, Mountain View, Calif.); the ALIGNprogram (Version 2.0), GAP, BESTFIT, BLAST, FASTA, Megalign (using JotunHein, Martinez, Needleman-Wunsch algorithms), DNAStar Lasergene (seewww.dnastar.com) and TFASTA in the Wisconsin Genetics Software Package,Version 8 (available from Genetics Computer Group (GCG), 575 ScienceDrive, Madison, Wis., USA). Alignments using these programs can beperformed using the default parameters or parameters selected by theoperator. The CLUSTAL program is well described by Higgins. The ALIGNprogram is based on the algorithm of Myers and Miller; and the BLASTprograms are based on the algorithm of Karlin and Altschul. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Typically, inhibition of target sequences by RNAi requires a high degreeof sequence homology between the target sequence and the sense strand ofthe RNAi molecules. In some embodiments, such homology is higher thanabout 70%, and may be higher than about 75%. Preferably, homology ishigher than about 80%, and is higher than 85% or even 90%. Morepreferably, sequence homology between the target sequence and the sensestrand of the RNAi is higher than about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99%. In embodiments where the multiple-promoter RNAiexpression construct is used to target viral infections, it may be thatsequence homology between the genomes of the various subspecies of thevirus, even in conserved regions, does not reach the level of over 90%or even 80% over 15 to 30 consecutive nucleotides. In such a case,sequence homology between the target sequence for some subspecies andthe sense strand of the RNAi may be 80% or less.

On the other hand, the multiple-promoter RNAi expression construct ofthe present invention is particularly useful when targeting genes oforganisms that do not display high sequence homology across species,subspecies or variants, as each RNAi species in the multiple promoterRNAi expression cassette can be used to address different portions ofthe target gene(s) or subsets of variants or subspecies.

In addition to selecting the RNAi sequences based on conserved regionsof a target sequence, selection of the RNAi sequences may be based onother factors. Despite a number of attempts to devise selection criteriafor identifying sequences that will be effective in RNAi based onfeatures of the desired target sequence (e.g., percent GC content,position from the translation start codon, or sequence similaritiesbased on an in silico sequence database search for homologs of theproposed RNAi, thermodynamic pairing criteria), it is presently notpossible to predict with much degree of confidence which of the myriadpossible candidate RNAi sequences correspond to a desired target will,in fact, elicit an RNA silencing response. Instead, individual specificcandidate RNAi polynucleotide sequences typically are generated andtested to determine whether interference with expression of a desiredtarget can be elicited.

A major problem of current anti-viral therapies is the emergence ofresistant variants, known generally as escape mutants (Gitlin et. al. J.of Virol. 79; 1027-1035, (2005)). One aspect of the present inventionneutralizes emergent escape mutants. In some embodiments of thisinvention the selection of multiple RNAi sequences to treat viralinfections is based on the emergence of escape mutants from treatment ofinfected cells with single sequence of RNAi. Emergent escape mutants aredetermined by treatment with an expression construct containing a singlesequence of RNAi after the cells have been infected with virus. Cellscontaining resistant viruses that emerge are harvested and the viralgenomes sequenced. Sequencing reveals predominant mutations that ariseto resist viral inhibition. A multiple-promoter RNAi expressionconstruct of the present invention is generated that contains RNAisequences based upon the genetic sequence of the target gene andadditionally sequences of the point mutations that arise to resist RNAitreatment.

As stated, the RNAi coding regions of the multiple-promoter RNAiexpression cassette are operatively linked to terminator elements. Inone embodiment, the terminators comprise stretches of four or morethymidine residues. In another preferred embodiment, the terminatorelements used are all different and are matched to the promoter elementsfrom the gene from which the terminator is derived. Such terminatorsinclude the SV40 poly A, the Ad VA1 gene, the 5S ribosomal RNA gene, andthe terminators for human t-RNAs. In addition, promoters and terminatorsmay be mixed and matched, as is commonly done with RNA pol II promotersand terminators.

In addition, the multiple-promoter RNAi expression cassettes may beconfigured where multiple cloning sites and/or unique restriction sitesare located strategically, such that promoter, RNAi and terminatorelements are easily removed or replaced. Moreover, the multiple-promoterRNAi expression cassettes may be assembled from smaller oligonucleotidecomponents using strategically located restriction sites and/orcomplementary sticky ends. The base vector for one approach according toembodiments of the present invention consists of plasmid with amultilinker in which all sites are unique (though this is not anabsolute requirement). Sequentially, each promoter is inserted betweenits designated unique sites resulting in a base cassette with threepromoters, or more, all of which can have variable orientation.Sequentially, again, annealed primer pairs are inserted into the uniquesites downstream of each of the individual promoters, resulting in atriple expression cassette construct. The insert can be moved into, e.g.an AAV backbone using two unique enzyme sites (the same or differentones) that flank the triple expression cassette insert.

In step 300 of FIG. 1, the multiple-promoter RNAi expression cassettesare ligated into a delivery vector. The constructs into which themultiple-promoter RNAi expression cassette is inserted and used for highefficiency transduction and expression of the RNAi agents in variouscell types preferably are derived from viruses and are compatible withviral delivery. Generation of the construct can be accomplished usingany suitable genetic engineering techniques well known in the art,including without limitation, the standard techniques of PCR,oligonucleotide synthesis, restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing. The constructpreferably comprises, for example, sequences necessary to package themultiple-promoter RNAi expression construct into viral particles and/orsequences that allow integration of the multiple promoter RNAiexpression construct into the target cell genome. The viral constructalso may contain genes that allow for replication and propagation ofvirus, though in preferred embodiments such genes will be supplied intrans. Additionally, the viral construct may contain genes or geneticsequences from the genome of any known organism incorporated in nativeform or modified. For example, the preferred viral construct comprisessequences useful for replication of the construct in bacteria.

The construct also may contain additional genetic elements. The types ofelements that may be included in the construct are not limited in anyway and may be chosen by one with skill in the art. For example,additional genetic elements may include a reporter gene, such as one ormore genes for a fluorescent marker protein such as GFP or RFP; aneasily assayed enzyme such as beta-galactosidase, luciferase,beta-glucuronidase, chloramphenical acetyl transferase or secretedembryonic alkaline phosphatase; or proteins for which immunoassays arereadily available such as hormones or cytokines. Other genetic elementsthat may find use in embodiments of the present invention include thosecoding for proteins which confer a selective growth advantage on cellssuch as adenosine deaminase, aminoglycodic phosphotransferase,dihydrofolate reductase, hygromycin-B-phosphotransferase, or thosecoding for proteins that provide a biosynthetic capability missing froman auxotroph. If a reporter gene is included along with themultiple-promoter RNAi expression cassette, an internal ribosomal entrysite (IRES) sequence can be included. Preferably, the additional geneticelements are operably linked with and controlled by an independentpromoter/enhancer.

A viral delivery system based on any appropriate virus may be used todeliver the multiple-promoter RNAi expression constructs of the presentinvention. In addition, hybrid viral systems may be of use. The choiceof viral delivery system will depend on various parameters, such as thetissue targeted for delivery, transduction efficiency of the system,pathogenicity, immunological and toxicity concerns, and the like. Giventhe diversity of disease targets that are amenable to interference bythe multiple-promoter RNAi expression constructs of the presentinvention, it is clear that there is no single viral system that issuitable for all applications. When selecting a viral delivery system touse in the present invention, it is important to choose a system wheremultiple-promoter RNAi expression construct-containing viral particlesare preferably: 1) reproducibly and stably propagated; 2) able to bepurified to high titers; and 3) able to mediate targeted delivery(delivery of the multiple-promoter RNAi expression construct to thetissue or organ of interest without widespread dissemination); 4) ableto be expressed in a constitutive or regulatable manner.

In general, the five most commonly used classes of viral systems used ingene therapy can be categorized into two groups according to whethertheir genomes integrate into host cellular chromatin (oncoretrovirusesand lentiviruses) or persist in the cell nucleus predominantly asextrachromosomal episomes (adeno-associated virus, adenoviruses andherpesviruses). This distinction is an important determinant of thesuitability of each vector for particular applications; non-integratingvectors can, under certain circumstances, mediate persistent geneexpression in non-proliferating cells, but integrating vectors are thetools of choice if stable genetic alteration needs to be maintained individing cells.

For example, in one embodiment of the present invention, viruses fromthe Parvoviridae family are utilized. The Parvoviridae is a family ofsmall single-stranded, non-enveloped DNA viruses with genomesapproximately 5000 nucleotides long. Included among the family membersis adeno-associated virus (AAV), a dependent parvovirus that bydefinition requires co-infection with another virus (typically anadenovirus or herpesvirus) to initiate and sustain a productiveinfectious cycle. In the absence of such a helper virus, AAV is stillcompetent to infect or transducer a target cell by receptor-mediatedbinding and internalization, penetrating the nucleus in bothnon-dividing and dividing cells.

Once in the nucleus, the virus uncoats and the transgene is expressedfrom a number of different forms—the most persistent of which arecircular monomers. AAV will integrate into the genome of 1-5% of cellsthat are stably transduced (Nakai, et al., J. Virol. 76:11343-349(2002). Expression of the transgene can be exceptionally stable and inone study with AAV delivery of Factor IX, a dog model continues toexpress therapeutic levels of the protein over 5.0 years after a singledirect infusion with the virus. Because progeny virus is not producedfrom AAV infection in the absence of helper virus, the extent oftransduction is restricted only to the initial cells that are infectedwith the virus. It is this feature which makes AAV a preferred genetherapy vector for the present invention. Furthermore, unlikeretrovirus, adenovirus, and herpes simplex virus, AAV appears to lackhuman pathogenicity and toxicity (Kay, et al., Nature. 424: 251 (2003)and Thomas, et al., Nature Reviews, Genetics 4:346-58 (2003)).

Typically, the genome of AAV contains only two genes. The “rep” genecodes for at least four separate proteins utilized in DNA replication.The “cap” gene product is spliced differentially to generate the threeproteins that comprise the capsid of the virus. When packaging thegenome into nascent virus, only the Inverted Terminal Repeats (ITRs) areobligate sequences; rep and cap can be deleted from the genome and bereplaced with heterologous sequences of choice. However, in orderproduce the proteins needed to replicate and package the AAV-basedheterologous construct into nascent virion, the rep and cap proteinsmust be provided in trans. The helper functions normally provided byco-infection with the helper virus, such as adenovirus or herpesvirusmentioned above also can be provided in trans in the form of one or moreDNA expression plasmids. Since the genome normally encodes only twogenes it is not surprising that, as a delivery vehicle, AAV is limitedby a packaging capacity of 4.5 single stranded kilobases (kb). However,although this size restriction may limit the genes that can be deliveredfor replacement gene therapies, it does not adversely affect thepackaging and expression of shorter sequences such as RNAi.

The utility of AAV for RNAi applications was demonstrated in experimentswhere AAV was used to deliver shRNA in vitro to inhibit p53 and Caspase8 expression (Tomar et al., Oncogene. 22: 5712-15 (2003)). Followingcloning of the appropriate sequences into a gutted AAV-2 vector,infectious AAV virions were generated in HEK293 cells and used to infectHeLa S3 cells. A dose-dependent decrease of endogenous Caspase 8 and p53levels was demonstrated. Boden et al. also used AAV to deliver shRNA invitro to inhibit HIV replication in tissue culture systems (Boden, etal., J. Virol. 77(21): 115231-35 (2003)) as assessed by p24 productionin the spent media.

However, technical hurdles must be addressed when using AAV as a vehiclefor multiple-promoter RNAi expression constructs. For example, variouspercentages of the human population may possess neutralizing antibodiesagainst certain AAV serotypes. However, since there are several AAVserotypes, some of which the percentage of individuals harboringneutralizing antibodies is vastly reduced, other serotypes can be usedor pseudo-typing may be employed. There are at least eight differentserotypes that have been characterized, with dozens of others which havebeen isolated but have been less well described. Another limitation isthat as a result of a possible immune response to AAV, AAV-based therapymay only be administered once; however, use of alternate, non-humanderived serotypes may allow for repeat administrations. Administrationroute, serotype, and composition of the delivered genome all influencetissue specificity.

Another limitation in using unmodified AAV systems with themultiple-promoter RNAi expression constructs is that transduction can beinefficient. Stable transduction in vivo may be limited to 5-10% ofcells. However, different methods are known in the art to boost stabletransduction levels. One approach is utilizing pseudotyping, where AAV-2genomes are packaged using cap proteins derived from other serotypes.For example, by substituting the AAV-5 cap gene for its AAV-2counterpart, Mingozzi et al. increased stable transduction toapproximately 15% of hepatocytes (Mingozzi, et al., J. Virol. 76(20):10497-502 (2002)). Thomas et al., transduced over 30% of mousehepatocytes in vivo using the AAV8 capsid gene (Thomas, et al., J.Virol. in press). Grimm et al. (Blood. 2003-02-0495) exhaustivelypseudotyped AAV-2 with AAV-1, AAV-3B, AAV-4, AAV-5, and AAV-6 for tissueculture studies. The highest levels of transgene expression were inducedby virion which had been pseudotyped with AAV-6; producing nearly 2000%higher transgene expression than AAV-2. Thus, the present inventioncontemplates use of a pseudotyped AAV virus to achieve high transductionlevels, with a corresponding increase in the expression of the RNAimultiple-promoter expression constructs.

Another viral delivery system useful with the multiple-promoter RNAiexpression constructs of the present invention is a system based onviruses from the family Retroviridae. Retroviruses comprisesingle-stranded RNA animal viruses that are characterized by two uniquefeatures. First, the genome of a retrovirus is diploid, consisting oftwo copies of the RNA. Second, this RNA is transcribed by thevirion-associated enzyme reverse transcriptase into double-stranded DNA.This double-stranded DNA or provirus can then integrate into the hostgenome and be passed from parent cell to progeny cells as astably-integrated component of the host genome.

In some embodiments, lentiviruses are the preferred members of theretrovirus family for use in the present invention. Lentivirus vectorsare often pseudotyped with vesicular steatites virus glycoprotein(VSV-G), and have been derived from the human immunodeficiency virus(HIV), the etiologic agent of the human acquired immunodeficiencysyndrome (AIDS); visan-maedi, which causes encephalitis (visna) orpneumonia in sheep; equine infectious anemia virus (EIAV), which causesautoimmune hemolytic anemia and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immunodeficiency virus (BIV) which causes lymphadenopathy andlymphocytosis in cattle; and simian immunodeficiency virus (SIV), whichcauses immune deficiency and encephalopathy in non-human primates.Vectors that are based on HIV generally retain <5% of the parentalgenome, and <25% of the genome is incorporated into packagingconstructs, which minimizes the possibility of the generation ofreverting replication-competent HIV. Biosafety has been furtherincreased by the development of self-inactivating vectors that containdeletions of the regulatory elements in the downstreamlong-terminal-repeat sequence, eliminating transcription of thepackaging signal that is required for vector mobilization.

Reverse transcription of the retroviral RNA genome occurs in thecytoplasm. Unlike C-type retroviruses, the lentiviral cDNA in complexeswith other viral factors—known as the pre-initiation complex—is able totranslocate across the nuclear membrane and transduce non-dividingcells. A structural feature of the viral cDNA—a DNA flap—seems tocontribute to efficient nuclear import. This flap is dependent on theintegrity of a central polypurine tract (cPPT) that is located in theviral polymerase gene, so most lentiviral-derived vectors retain thissequence. Lentiviruses have broad tropism, low inflammatory potential,and result in an integrated vector. The main limitations are thatintegration might induce oncogenesis in some applications. The mainadvantage to the use of lentiviral vectors is that gene transfer ispersistent in most tissues or cell types.

A lentiviral-based construct used to express the RNAi agents preferablycomprises sequences from the 5′ and 3′ long terminal repeats (LTRs) of alentivirus. More preferably the viral construct comprises an inactivatedor self-inactivating 3′ LTR from a lentivirus. The 3′ LTR may be madeself-inactivating by any method known in the art. In a preferredembodiment, the U3 element of the 3′ LTR contains a deletion of itsenhancer sequence, preferably the TATA box, Sp1 and NF-kappa B sites. Asa result of the self-inactivating 3′ LTR, the provirus that isintegrated into the host genome will comprise an inactivated 5′ LTR. TheLTR sequences may be LTR sequences from any lentivirus from any species.The lentiviral-based construct also may incorporate sequences for MMLVor MSCV, RSV or mammalian genes. In addition, the U3 sequence from thelentiviral 5′ LTR may be replaced with a promoter sequence in the viralconstruct. This may increase the titer of virus recovered from thepackaging cell line. An enhancer sequence may also be included.

Other viral or non-viral systems known to those skilled in the art maybe used to deliver the multiple-promoter RNAi expression cassettes ofthe present invention to cells of interest, including but not limited togene-deleted adenovirus-transposon vectors that stably maintainvirus-encoded transgenes in vivo through integration into host cells(see Yant, et al., Nature Biotech. 20:999-1004 (2002)); systems derivedfrom Sindbis virus or Semliki forest virus (see Perri, et al, J. Virol.74(20):9802-07 (2002)); systems derived from Newcastle disease virus orSendai virus; or mini-circle DNA vectors devoid of bacterial DNAsequences (see Chen, et al., Molecular Therapy. 8(3):495-500 (2003)).Mini-circle DNA as described in U.S. Publ. No. 2004/0214329 disclosesvectors that provide for persistently high levels of nucleic acidtranscription. The circular vectors are characterized by being devoid ofexpression-silencing bacterial sequences, and may include aunidirectional site-specific recombination product sequence in additionto an expression cassette.

In addition, hybrid viral systems may be used to combine usefulproperties of two or more viral systems. For example, the site-specificintegration machinery of wild-type AAV may be coupled with the efficientinternalization and nuclear targeting properties of adenovirus. AAV inthe presence of adenovirus or herpesvirus undergoes a productivereplication cycle; however, the in the absence of helper functions, theAAV genome integrates into a specific site on chromosome 19. Integrationof the AAV genome requires expression of the AAV rep protein. Asconventional AAV vectors are deleted for all viral genes including rep,they are not able to specifically integrate into chromosome 19. However,this feature may be exploited in an appropriate hybrid system. Inaddition, non-viral genetic elements may be used to achieve desiredproperties in a viral delivery system, such as genetic elements thatallow for site-specific recombination.

In step 400 of FIG. 1, the multi-promoter RNAi expression construct ispackaged into viral particles. Any method known in the art may be usedto produce infectious viral particles whose genome comprises a copy ofthe viral multiple-promoter RNAi expression construct. FIGS. 4A and 4Bshow alternative methods for packaging the multiple-promoter RNAiexpression constructs of the present invention into viral particles fordelivery. The method in FIG. 4A utilizes packaging cells that stablyexpress in trans the viral proteins that are required for theincorporation of the viral multiple-promoter RNAi expression constructinto viral particles, as well as other sequences necessary or preferredfor a particular viral delivery system (for example, sequences neededfor replication, structural proteins and viral assembly) and eitherviral-derived or artificial ligands for tissue entry. In FIG. 4A, amultiple-promoter RNAi expression cassette is ligated to a viraldelivery vector (step 300), and the resulting viral multiple-promoterRNAi expression construct is used to transfect packaging cells (step410). The packaging cells then replicate viral sequences, express viralproteins and package the viral multiple-promoter RNAi expressionconstructs into infectious viral particles (step 420). The packagingcell line may be any cell line that is capable of expressing viralproteins, including but not limited to 293, HeLa, A549, PerC6, D17,MDCK, BHK, bing cherry, phoenix, Cf2Th, or any other line known to ordeveloped by those skilled in the art. One packaging cell line isdescribed, for example, in U.S. Pat. No. 6,218,181.

Alternatively, a cell line that does not stably express necessary viralproteins may be co-transfected with two or more constructs to achieveefficient production of functional particles. One of the constructscomprises the viral multiple-promoter RNAi expression construct, and theother plasmid(s) comprises nucleic acids encoding the proteins necessaryto allow the cells to produce functional virus (replication andpackaging construct) as well as other helper functions. The method shownin FIG. 4B utilizes cells for packaging that do not stably express viralreplication and packaging genes. In this case, the promoter RNAiexpression construct is ligated to the viral delivery vector (step 300)and then co-transfected with one or more vectors that express the viralsequences necessary for replication and production of infectious viralparticles (step 430). The cells replicate viral sequences, express viralproteins and package the viral multiple-promoter RNAi expressionconstructs into infectious viral particles (step 420).

The packaging cell line or replication and packaging construct may notexpress envelope gene products. In these embodiments, the gene encodingthe envelope gene can be provided on a separate construct that isco-transfected with the viral multiple-promoter RNAi expressionconstruct. As the envelope protein is responsible, in part, for the hostrange of the viral particles, the viruses may be pseudotyped. Asdescribed supra, a “pseudotyped” virus is a viral particle having anenvelope protein that is from a virus other than the virus from whichthe genome is derived. One with skill in the art can choose anappropriate pseudotype for the viral delivery system used and cell to betargeted. In addition to conferring a specific host range, a chosenpseudotype may permit the virus to be concentrated to a very high titer.Viruses alternatively can be pseudotyped with ecotropic envelopeproteins that limit infection to a specific species (e.g., ecotropicenvelopes allow infection of, e.g., murine cells only, where amphotropicenvelopes allow infection of, e.g., both human and murine cells.) Inaddition, genetically-modified ligands can be used for cell-specifictargeting, such as the asialoglycoprotein for hepatocytes, ortransferrin for receptor-mediated binding.

After production in a packaging cell line, the viral particlescontaining the multiple-promoter RNAi expression cassettes are purifiedand quantified (titered). Purification strategies include densitygradient centrifugation, or, preferably, column chromatographic methods.

The viral multiple-promoter RNAi expression cassettes of the presentinvention are particularly useful as therapeutics to treat disease or asvaccines to prevent disease. For example, the multiple-promoter RNAiexpression constructs may be introduced into a cancerous cell or tumorto inhibit expression of a gene required for maintenance of thecarcinogenic/tumorigenic phenotype. Similarly, the multiple-promoterRNAi expression constructs may be introduced into a cell infected with apathogen such as a virus to inhibit gene expression of one or more genesrequired for maintenance of the pathogen. To prevent a disease or otherpathology, a multiple-promoter RNAi expression construct may be used asa vaccine to target a gene required for initiation or maintenance of thedisease or pathology.

The viral multiple-promoter RNAi expression constructs of the presentinvention may be used in the treatment of cancer, including solid tumorsand leukemias, including: apudoma, choristoma, branchioma, malignantcarcinoid syndrome, carcinoid heart disease, carcinoma (e.g., Walker,basal cell, basosquamous, Brown-Pearce, ductal, Ehrlich tumor, in situ,Krebs 2, Merkel cell, mucinous, non-small cell lung, oat cell,papillary, scirrhous, bronchiolar, bronchogenic, squamous cell, andtransitional cell), histiocytic disorders, leukemia (e.g., B cell, mixedcell, null cell, T cell, T-cell chronic, HTLV-II-associated, lymphocyticacute, lymphocytic chronic, mast cell, and myeloid), hystiocytosismalignant, Hodgkin disease, immunoproliferative small, non-Hodgkinlymphoma, plasmacytoma, reticuloendotheliosis, melanoma,chondroblastoma, chondroma, chondrosarcoma, fibroma, fibrosarcoma, giantcell tumors, histiocytoma, lipoma, liposarcoma, mesothelioma, myxoma,myxosarcoma, osteoma, osteosarcoma, Ewing sarcoma, synovioma,adenofibroma, adenolymphoma, carcinosarcoma, chordoma,cranio-pharyngioma, dysgerminoma, hamartoma, mesenchymoma, mesonephroma,myosarcoma, ameloblastoma, cementoma, odontoma, teratoma, thymoma,trophoblastic tumor, adenocarcinoma, adenoma, cholangioma,cholesteatoma, cylindroma, cystadenocarcinoma, cystadenoma, granulosacell tumor, gynandroblastoma, hepatoma, hidradenoma, islet cell tumor,Leydig cell tumor, papilloma, Sertoli cell tumor, theca cell tumor,leiomyoma, leiomyosarcoma, myoblastoma, myoma, myosarcoma, rhabdomyoma,rhabdomyosarcoma, ependymoma, ganglioneuroma, glioma, medulloblastoma,meningioma, neurilemmoma, neuroblastoma, neuroepithelioma, neurofibroma,neuroma, paraganglioma, paraganglioma nonchromaffin, angiokeratoma,angiolymphoid hyperplasia with eosinophilia, angioma sclerosing,angiomatosis, glomangioma, hemangioendothelioma, hemangioma,hemangiopericytoma, hemangiosarcoma, lymphangioma, lymphangiomyoma,lymphangiosarcoma, pheochromocytoma, pinealoma, carcinosarcoma,chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma, hemangiosarcoma,leiomyosarcoma, leukosarcoma, liposarcoma, lymphangiosarcoma,myosarcoma, myxosarcoma, ovarian carcinoma, rhabdomyosarcoma, sarcoma(e.g., Ewing, experimental, Kaposi, and mast cell), neoplasms (e.g.,bone, breast, digestive system, colorectal, liver, pancreatic,pituitary, testicular, orbital, head and neck, central nervous system,acoustic, pelvic, respiratory tract, and urogenital), neurofibromatosis,and cervical dysplasia, and for treatment of other conditions in whichcells have become immortalized or transformed. In addition, themultiple-promoter RNAi expression constructs of the present inventioncould be used in combination with other treatment modalities, such aschemotherapy, surgical intervention, cryotherapy, hyperthermia,radiation therapy, and the like.

A gene involved in the replication of a pathogen, transmission of apathogen, or maintenance of infection may be targeted by the viralmultiple-promoter RNAi expression constructs. Such viralmultiple-promoter RNAi expression constructs may be used to treat cellsat risk for infection by a pathogen (i.e., vaccine) or cells thatalready have been infected. Pathogens that may be treated by themultiple-promoter RNAi expression constructs and methods of the presentinvention include viruses from the families Parvoviridae, Papovaviridae(including Papilloma virus, etc.), Adenoviridae, Herpesviridae(including herpesvirus types 1 through 7), Poxviridae, Hepadnaviridae,Picornaviridae (coxsackie A and coxsackie B viruses and ECHOvirus),Caliciviridae, Reoviridae, Togaviridae (encephalitis viruses),Flaviviridae (encephalitis viruses), Arenaviridae, Retroviridae,Bunyaviridae, Coronaviridae, Orthomyzoviridae, Paramyxoviridae,Rhabdoviridae and Filoviridae, bacteria generally, mycobacteria, fungi,Falciparum, Tryponosoma Schistosoma, and the like.

In step 500 of FIG. 1, the multiple-promoter RNAi expression constructis delivered to the cells to be treated. The multiple-promoter RNAiexpression construct of the present invention may be introduced into thecells in vitro or ex vivo and then subsequently placed into an animal toaffect therapy, or administered directly to an organism, organ or cellby in vivo administration. Delivery by viral infection is a preferredmethod of delivery; however, any appropriate method of delivery of themultiple-promoter RNAi expression construct may be employed. The vectorscomprising the multiple-promoter cassettes can be administered to amammalian host using any convenient protocol, where a number ofdifferent such protocols are known in the art.

The nucleic acids may be introduced into tissues or host cells by anynumber of routes, including viral infection, microinjection, or fusionof vesicles. Injection may also be used for intra-muscularadministration, as described by Furth et al., Anal. Biochem.115(205):365-368 (1992). The nucleic acids may be coated onto goldmicroparticles, and delivered intradermally by a particle bombardmentdevice, or “gene gun” as described in the literature (see, for example,Tang et al., Nature. 356:152-154 (1992)), where gold microprojectilesare coated with the DNA, then bombarded into skin cells.

Another delivery method useful for the method of the present inventioncomprises the use of Cyclosert™ technology as described in U.S. Pat. No.6,509,323 to Davis et.al. Cyclosert™ technology platform is based uponcup-shaped cyclic repeating molecules of glucose known as cyclodextrins.The “cup” of the cyclodextrin molecule can form “inclusion complexes”with other molecules, making it possible to combine the Cyclosert™polymers with other moieties to enhance stability or to add targetingligands. In addition, cyclodextrins generally have been found to be safein humans (individual cyclodextrins currently enhance solubility inFDA-approved oral and IV drugs) and can be purchased in pharmaceuticalgrade on a large scale at low cost. These polymers are extremely watersoluble, non-toxic and non-immunogenic at therapeutic doses, even whenadministered repeatedly. The polymers can easily be adapted to carry awide range of small-molecule therapeutics at drug loadings that can besignificantly higher than liposomes.

The vectors comprising the multiple-promoter cassettes can be formulatedinto preparations for injection or administration by dissolving,suspending or emulsifying them in an aqueous or nonaqueous solvent, suchas oils, synthetic aliphatic acid glycerides, esters of higher aliphaticacids or propylene glycol; and if desired, with conventional additivessuch as solubilizers, isotonic agents, suspending agents, emulsifyingagents, stabilizers and preservatives.

In addition, the vectors comprising the multiple-promoter cassettes canbe formulated into pharmaceutical compositions by combination withappropriate, pharmaceutically acceptable carriers or diluents. Inpharmaceutical dosage forms, the vectors comprising themultiple-promoter cassettes may be administered alone or in associationor combination with other pharmaceutically active compounds. Those withskill in the art will appreciate readily that dose levels for vectorscomprising the multiple-promoter cassettes will vary as a function ofthe nature of the delivery vehicle, the relative ease of transduction ofthe target cells, the expression level of the RNAi species in the targetcells and the like.

EXAMPLES

One disease state that may be treated with the multiple-promoter RNAiexpression constructs of the present invention is hepatitis C virus(HCV) infection. Based on statistics compiled from the Centers forDisease Control and Prevention, almost 2% of the Americanpopulation—nearly 4 million people—is currently infected with HCV.Initially, the majority of the individuals infected with HCV exhibit nosymptoms; however, greater than 80% will develop chronic and progressiveliver disease eventually leading to cirrhosis or hepatocellularcarcinomas. HCV is the leading indication for liver transplantationwithin the United States and results in the death of 8,000 to 10,000Americans every year. On a global level, the World Heath Organizationestimates that there are more than 170 million affected individuals,with infection rates as high as 10-30% of the general population in somecountries.

HCV is a positive-sense single stranded enveloped RNA virus belonging tothe Flaviviridae family. The infectious cycle of HCV typically beginswith the entry of the viral particle into the cell by receptor-mediatedbinding and internalization. After uncoating in the cytoplasm, thepositive strand of RNA that comprises the genome can interact directlywith the host cell translational machinery. Lacking 5′ cap methylation,the RNA forms an extensive secondary structure in the 5′ UntranslatedRegion (UTR) that serves as an internal ribosomal entry site (IRES) andpermits the direct binding of the 40S subunit as the initiating step ofthe translation process.

The HCV genome, approximately 9600 nucleotides in length, encodes asingle long open reading frame termed the polyprotein (shown in FIG.8A). Viral proteins are produced as linked precursors from thepolyprotein which is subsequently cleaved into mature products by a widevariety of viral and cellular enzymes. Encoded amongst the genes are thestructural proteins, including the core and envelope glycoproteins, sonamed because they are integral structural components in progenyvirions. Non-structural proteins, which provide indispensable functionssuch as the RNA-dependent RNA polymerase, are also produced. The viralreplication machinery is established within the cytoplasm of infectedcells that transcribe the positive-sense RNA into a negative strandintermediate. Thus, the HCV genomic RNA serves as both a template forits own replication and as a messenger RNA for translation of thevirally encoded proteins. The negative strand is transcribed back into apositive strand of RNA, thereby amplifying the number of positive strandcopies within the cell. At this stage, the positive strand can interactwith the host cell translational machinery once again or, if there havebeen enough structural proteins accumulated, be packaged into virions.Following egress from the cell, the virus repeats its infectious cycle.

Example 1 Development of an AAV-2 Expression Vector For in vivo Deliveryof shRNA Sequences

Before the delivery of shRNA by infectious particles is tested, theappropriate expression plasmid is constructed and validated. There areat least two characteristics that can be considered when designing themultiple-promoter RNAi expression construct: 1) the construct must beefficiently packaged into progeny virion; and 2) the plasmid mustprovide high levels of shRNA expression. In addition, in order to testthe various multiple-promoter RNAi expression constructs, there must bea means of assessing transfection and transduction efficiency.

AAV-2 vectors which have been gutted of rep and cap sequence provide thebackbone (hereinafter referred to as the rAAV vector) for the viral RNAiexpression construct. This vector has been extensively employed in AAVstudies and the requirements for efficient packaging are wellunderstood. The U6 and H1 promoters are used for the expression of shRNAsequences, though there have been reports of vastly different levels ofinhibition of an identical shRNA driven independently by each promoter.However, vector construction is such that promoters can be easilyswapped if such variation is seen.

As with virtually any viral delivery system, the rAAV vector must meetcertain size criteria in order to be packaged efficiently. In general,an rAAV vector must be 4300-4900 nucleotides in length (McCarty, et al.Gene Ther. 8: 1248-1254 (2001)). When the rAAV vector falls below thelimit, a ‘stuffer’ fragment must be added (Muzyczka, et al. Curr. Top.Microbiol. Immunol. 158: 970129 (1992)). Alternatively, the rAAVmultiple-promoter RNAi expression construct may be filled out with twoor more multiple-promoter RNAi expression cassettes.

In the rAAV vector embodiment described here, eachpromoter/RNAi/terminator component is approximately 400 nucleotides inlength, leaving ample room for the inclusion of manypromoter/RNAi/terminator components per expression cassette.Alternatively, one or more selectable marker cassettes may be engineeredinto the rAAV multiple-promoter RNAi expression construct in order toassess the transfection efficiency of the rAAV expression construct aswell as allow for quantification of transduction efficiency of targetcells by the rAAV expression construct delivered via infectiousparticles.

The initial test expression construct drives expression of a shRNAspecies designed from sequences with demonstrated ability to inhibitluciferase activity from a reporter construct (See, Elbashir, et al.Embo. J. 20(23): 6877-6888 (2001)). The elements of the RNAi cassette,including the promoter, shRNA and the terminator sequence, are short andare assembled independently de novo utilizing long, complementaryoligonucleotides that are then cloned into a viral vector using multiplecloning sites. A commercially available expression plasmid that encodesfor the production of luciferase functions as the reporter in order toverify the ability of the shRNA to down regulate the target sequences(as shown in FIG. 5).

Although the shRNA against luciferase has been previously validated, theefficacy of rAAV-delivered shRNA is assessed in vitro prior to testingthe construct in vivo. The test and reporter constructs are transfectedinto permissive cells utilizing standard techniques. An rAAV expressionconstruct in which the luciferase-specific shRNA has been replaced by anunrelated shRNA sequence is utilized as a negative control in theexperiments. The relative percentage of transfection efficiency isestimated directly by assessing the levels of the selective marker usingfluorescence microscopy. For assessing inhibitory activity of the shRNA,luciferase activity is measured utilizing standard commercial kits.Alternatively, quantitative real time PCR analysis (Q-PCR) is run on RNAthat is harvested and purified from parallel experimental plates.Activity decreases greater than 90% percent, relative to the activityrecovered in lysates from cells treated with the unrelated shRNAspecies, are an indication that the shRNA is highly functional.

Subsequent experiments are performed in order to assess the effects ofshRNA on a luciferase reporter system that is transfected into thelivers of mice, similar to the work of McCaffrey et al. in Nature. 418:38-39 (2002). Nucleic acids delivered to mice by hydrodynamictransfection methods (high pressure tail vein injection) primarilylocalized to the livers. Much like the principle which governsco-transfection in cell culture, simultaneous injection of multipleplasmids from a mixture often permits the penetration of all of theexpression constructs into the same cell. Thus, even though the tailvein injection procedures are well documented to only transfect 5-40% ofthe hepatocytes within the liver (McCaffrey, et al. Nature Biotech.21(6): 639-644 (2003)), co-injection permits delivery of the reportersystem and the expression construct into the same cells.

The rAAV expression construct bearing the shRNA sequence targetedagainst luciferase is co-injected with the reporter construct thatencodes for the luciferase gene. In animals receiving the negativecontrol, an expression construct bearing an unrelated shRNA isco-injected with the reporter construct. After seven days, the mice aresacrificed and the livers harvested. Luciferase activity is measured onlysates generated from a portion of the liver. Remaining portions of theliver are utilized for Q-PCR measurements as well as histologicalanalysis to determine marker protein expression for normalization of thedata. Alternative methods to assess transfection efficiency may includeELISA measurements of serum from mice that have been co-injected with athird marker plasmid for a secreted protein such as human α1-antitrypsin(hAAT) (Yant, et al. Nature Genetics. 25: 35-41 (2000), see alsoMcCaffrey, et al. Nature Biotech. 21(6): 639-644 (2003)).

Once it is established that the expression construct is functional inboth in vitro cell culture systems as well as in vivo mouse models byutilizing co-transfection of the naked DNA plasmids, testing isinitiated on the rAAV expression construct packaged into infectiousparticles. The infectious particles are produced from a commerciallyavailable AAV helper-free system that requires the co-transfection ofthree separate expression constructs containing 1) the rAAV constructexpressing the shRNA against luciferase (flanked by the AAV ITRs); 2)the construct encoding the AAV rep and cap genes; and 3) an expressionconstruct comprising the helper adenovirus genes required for theproduction of high titer virus. Following standard purificationprocedures, the viral particles are ready for use in experiments.

Before mice can be infused with the rAAV particles, a reporter system isestablished in the mouse livers. Hydrodynamic transfection is employedto deliver the luciferase reporter construct as well as an expressionplasmid for hAAT to control for differences in transfection efficienciesfrom animal to animal. The mice are permitted to recover for severaldays in order to establish sufficient levels of reporter activity.

After luciferase reporter activity has been established in the livers,rAAV particles are infused into normal C57B1/6 mice either throughportal vein or tail vein injection. rAAV particles bearing theexpression construct of an unrelated shRNA are used as a negativecontrol. Initially, the mice are infused with relatively high doses(2×10¹² vector genomes (vg)) which are reduced in follow-up experimentsperformed to generate dose-response curves. After seven to ten days, themice are sacrificed, the livers harvested and samples of serumcollected. The relative levels of hepatic luciferase activity and RNAare determined from the isolated livers utilizing the luciferase assayand QPCR procedures previously described. Additionally, the efficiencyof transduction is assessed by measurement of the marker protein inserial slices of the hepatic tissues.

Results from the experiment may be wide ranging. It has been estimatedthat hydrodynamic transfection procedures may result in the transfectionof 5-40% of hepatocytes. Transduction of liver cells by AAV-2 deliveryprocedures have been shown to result in 5-10% transduction efficiencies.Although AAV may preferentially transduce the same pool of hepatocytesthat were transfected by the initial tail vein injection procedure, itis possible that the subsets of cells that each technique affects arenon-overlapping. If the former occurs, a reduction in luciferaseactivity relative to mice transduced with an unrelated shRNA species isseen. If the latter occurs, then no decrease in luciferase activity isseen.

It must be verified that AAV particles delivered by the tripleexpression construct inhibit the luciferase-HCV fusion reporter invitro. Permissive tissue culture cells are transfected with one of thereporter constructs detailed in FIG. 9. In addition, eachco-transfection mixture is supplemented with a plasmid coding for hAAT.Following 48 hours of incubation, cells are dosed with infectiousparticles harboring the triple promoter shRNA expression plasmid againstHCV. AAV particles containing a triple promoter construct expressingthree unrelated shRNA species serve as the negative control. Measurementof luciferase activity is used to verify that the AAV-delivered shRNAare highly functional.

Example 2 Modifications to Enhance Efficiency of AAV Transduction ofLiver Tissues

Although it has been demonstrated that AAV-based vectors can deliverdesired sequences to hepatocytes, the relative level of transductionthat occurs within those tissues has traditionally been rather poor. Forcurrent clinical hemophilia studies which employ AAV-2 to deliver andexpress blood factor IX, this is not a significant issue. For treatmentof hemophilia, it is critical only to replenish levels of secretedprotein to therapeutic levels. Such replenishment may occur from a smallnumber of transduced cells able to express significant levels of thedesired protein. However, because the mechanism of RNAi action isintracellular and the effect is not transmitted directly from cell tocell, the transduction efficiency must be increased in order for AAVexpressing shRNA to be utilized as a therapeutic.

McCarty et al. were able to generate a self complementary AAV vector(scAAV) that has both a plus and a minus strand of the same expressioncassette within its capsid Gene Ther. 8: 1248-1254 (2001)). This wasachieved by mutating the 5′ ITR and leaving the 3′ ITR intact. Bymutating or deleting the terminal resolution site other non-essentialAAV sequences, thus eliminating possible recombination by wild type AAVand this construct, a DNA template is created where replication startsat the 3′ ITR. Once the replication machinery reaches the 5′ ITR, noresolution takes place and replication continues to the 3′ ITR. Theresulting product has both a plus and complementary minus strand, yet isefficiently packaged. Employing the scAAV vectors, transduction of livercells was increased to 30% of the total hepatocytes (Fu, et al. MolecTherapy. 8(6):911-7. (2003)). When delivered intercisternally, more than50% of the Purkinje cells in the cerebellum were transduced by the scAAVparticles. Thomas et al. showed that self-complementary vectors couldproduce 50-fold higher luciferase transgene expression levels in mouselivers than their corresponding single-stranded AAV counterparts wheninfused into mouse livers at equivalent doses (Thomas, et al., J. Virol.(in press)). Though dropping slightly, the relative difference ofexpression between the vectors persisted at 20-fold nearly one yearafter injection.

Similar strategies are employed herein. Because of the size limitationsassociated with packaging the rAAV multiple-promoter RNAi expressionconstruct, the amount of space that may be utilized for delivery oftherapeutic sequences—already quite small by comparison to other viraldelivery systems—is halved by the use of scAAV. Thus, instead of beingable to package 4500 nucleotides, the limit is lowered to 2250nucleotides. Regardless, the size of the multiple-promoter RNAiexpression cassette allows such construction. A graphic of the majorelements within the scAAV is shown in FIG. 6.

Other modifications of AAV-delivery systems also have been used todramatically enhance transduction efficiencies, including the productionof pseudotyped viral particles by packaging rAAV-2 vector genomes withthe Cap protein from other serotypes. Because they have been among thebest characterized of all of the serotypes, the Cap proteins from AAV-1through AAV-6 are used most commonly to pseudotype the AAV-2 vectors.Even with the advantages gained by these employing pseudotypingstrategies, the threshold of transduction efficiency of hepatocytes maybe increased only to 15% of the total population. However, dozens ofother serotypes of AAV have been isolated and identified, but have notbeen characterized to any appreciable degree. For example, one of theseis AAV-8, which was isolated originally from the heart tissue of arhesus monkey. In an effort to determine effects novel cap proteins ontransduction, pseudotyped virus in which the single stranded AAV-2genome was pseudotyped with AAV-8 cap was created. The vectors carriedthe LacZ gene to assess the relative efficiency of transduction of mouselivers after infusion with increasing doses of infectious particles. Asummary of the results (Thomas, et al. J Virol. 78(6):3110-22. (2004))is shown below in Table 1:

TABLE 1 AAV-2/2 and AAV-2/8 Dose Response (% beta-gal positivehepatocytes) DOSE (v.g./Mouse) Vector 5 × 10¹⁰ 3 × 10¹⁰ 1.8 × 10¹¹ 3.9 ×10¹² 7.2 × 10¹² AAV-2/2 0.6 ± 0.4%  3.0 ± 0.5%  8.1 ± 1.0% 8.9 ± 1.0% NALacZ AAV-2/8 8.1 ± 1.8% 14.9 ± 3.4% 65.8 ± 9.1% NA 97.4 ± 0.3% LacZ

As the dose of infused control AAV-2/2 particles is increased, there isa modest increase in transduction of hepatocytes; however, the upperthreshold of transduction remains entrenched near the 10% limit.Surprisingly, pseudotyped AAV-2/8 particles transduced 8% of hepatocytesat the lowest dose of particles administered; doses that were 30-80 foldless than their AAV-2/2 counterparts. Additionally, the dose-dependentincrease in transduction efficiency for AAV-2/8 surpassed thetransduction efficiency for AAV-2/2 to greater than 97% at the highestdose. Transduction efficiencies within this range enable to efficientdelivery of RNAi to cells within tissues.

Similar modifications of AAV are engineered into the rAAV RNAiexpression constructs. Following incorporation of these simplemodifications, stocks of virus are generated for testing in the mousemodel system. The following rAAV RNAi experimental virus stocks aretested: single-strand AAV-2/2; single-strand AAV-2/8; self-complementaryAAV-2/2; and self-complementary AAV-2/8.

Corresponding viral particles that harbor rAAV vectors expressingunrelated shRNA sequences are produced and used as negative controls.Large decreases in relative levels of luciferase activity correlate withincreases in transduction efficiency.

Example 3A Selection and Testing of RNAi Agents Against a Luciferase-HCVFusion Plasmid

The selection of shRNAs useful as therapeutics against HCV is not astraight-forward proposition. In addition to the problem of thegeneration of escape mutants, the high mutation rate leads to a ratherlarge degree of sequence divergence within a population of infectedindividuals harboring the virus, with genotypes differing by as much as31-34% in their nucleotide sequences. Subtypes (species within a givengenotype) may differ by 20-23% based on full-length genomic sequencecomparisons. Thus, regions of the viral genome with a high degree ofconservation preferably are identified and chosen to ensure the broadesttherapeutic applicability. As an example of how sequences are alignedand therapeutically relevant regions selected, 30 full-length sequencescorresponding to HCV genotype 1b virus were retrieved from publicdatabases and aligned using the Jotun Hein Method and MegAlign analysissoftware (DNASTAR). Regions with a high degree of conservation wereidentified, such as a region in the 5′ UTR (nucleotides 75-112).

To select candidate sequences, an alignment of all published independentfull-length or near-full-length HCV sequences was performed; currentlythere are over 200 such sequences available representing all knowngenotypes. Several candidate regions for selection and development ofRNAi therapeutics currently exist and it is well-documented that the 5′and 3′ UTR regions are amongst the most highly conserved regions in theHCV genome. Despite perception that these non-coding sequences may notrepresent optimal sequences to target due to the potential for sterichindrance with the cellular translation complex proteins or regulatoryproteins, Yokota et al. have already identified a highly functional RNAitargeting the 5′ UTR in a replicon system (EMBO Rep. 4(6): 602-608(2003)). Although it would be beneficial to identify several regions ofabsolute identity within individual stretches of 21 nucleotides (thecorresponding size of the targeting sequences in a shRNA species),analysis to date demonstrates that such a degree of conservation doesnot occur within the various subtypes of a specified genotype, let aloneacross all genotypes. Thus, selection may include segments of the genomein which greater than 80% of the regions maintain absolute conservation.In a final construction of a preclinical candidate, the expression ofthree independent shRNAs compensates for the sequence variability,allowing for a combination therapy contained within a single deliveryvehicle.

Alternatively, if conserved regions that meet the selection criterion inan analysis of all HCV genotypes are not identified, sequence analysismay be restricted to genotype 1 (1a and 1b), which accounts for nearlythree quarters of the infected population with the United States and,with the exception of Africa, is the predominate genotype throughout theworld. In addition, the most current effective anti-HCV therapy, acombination of pegylated interferon with Ribavirin (a guanosineanalogue), is rather inefficient against genotype 1, but highlyefficient against the other genotypes. Thus, the greatest need for analternative therapy exists in the largest patient population. Assequence alignments only reveal homology, other selection criteria, suchas relative GC content and the lack of cross specificity when queriedagainst sequence databases, is applied when selecting the final RNAiagents to be tested.

For example, for one experiment, alignment was performed for multiplesequences from HCV subtypes 1a and 1b. A few conserved regions wereidentified as being long enough from which to select RNAi agents fortesting (>19 nucleotides). The 5′UTR and 3′UTR regions were the mostconserved regions. Since the regions of homology that were identifiedwere quite long, alignment also was performed between differentgenotypes. Combining the two alignments allowed selection of universallyconserved regions. Some regions, such as a long stretch of A's or U's,or of G's and C's were removed from consideration because they are notamenable to targeting with RNAi agents, leaving “qualified” regions forfurther selection. Only one universally conserved region was identifiedin the whole coding region (the open reading frame) for all of thegenotypes of HCV considered; therefore, the sequences selected fortargets in most cases were those that are conserved in subtypes 1a and1b.

Once “qualified” regions were identified, individual RNAi sequences wereselected applying the criterion that the 5′ end of the antisense strandin the RNAi agent should possess a lower free energy than the 3′ end.“Neighbor pair free energy” rules were applied to calculate free energyfor the terminal five nucleotides on both the 5′ and 3′ ends of allpotential RNAi agents selected thus far. As a result, a total of 30potential RNAi agents were identified: ten in the 5′UTR (5′-n), twelvein the Open Reading Frame ORF (C-n), and eight in the 3′UTR (3′-n) (seeTable 2). The relative locations of these RNAi target sites on the HCVgenome are shown in FIG. 8A.

TABLE 2 RNAi Sequences RNAi RNAi Luc-HCV Reporter HCV agent Sequence^(‡)SEQ ID NO. Plasmid Location 5′-1 gCTGTGAGGAACTACTGTCT SEQ ID NO. 1 20IRES 43-62 5′-2 GTCTAGCCATGGCGTTAGT SEQ ID NO. 2 — IRES 77-95 5′-3GGAGAGCCATAGTGGTCTG SEQ ID NO. 3 16, 20 IRES 131-149 5′-4GCGGAACCGGTGAGTACAC SEQ ID NO. 4 16 IRES 150-168 5′-5GTCTGCGGAACCGGTGAGTA SEQ ID NO. 5 16 IRES 146-165 5′-6GCGAAAGGCCTTGTGGTACT SEQ ID NO. 6 16, 17 IRES 270-289 5′-7GATAGGGTGCTTGCGAGTG SEQ ID NO. 7 16 IRES 295-313 5′-8GAGGTCTCGTAGACCGTGCA SEQ ID NO. 8 16, 17 IRES 319-338 5′-9gCTTGTGGTACTGCCTGATA SEQ ID NO. 9 — IRES 279-298  5′-10gCTGCCTGATAGGGTGCTTG SEQ ID NO. 10 17 IRES 289-307 C-1AGATCGTTGGTGGAGTTTA SEQ ID NO. 11 — Core 427-445 C-2gTTGGGTAAGGTCATCGATA SEQ ID NO. 12 — Core 696-714 C-3GCCGACCTCATGGGGTACAT SEQ ID NO. 13 18 Core 732-752 C-4GGTTGCTCTTTCTCTATCT SEQ ID NO. 14 — Core 852-870 C-5 GGGATATGATGATGAACTGSEQ ID NO. 15 — NS1 1300-1318 C-6 GGATGAACCGGCTAATAGC SEQ ID NO. 16 —NS4B 6085-6113 C-7 GGAGATGGGCGGCAACATC SEQ ID NO. 17 — NS5A 7046-7064C-8 GTCTTCACGGAGGCTATGA SEQ ID NO. 18 — NS5B 8610-8629 C-9GTCAACTCCTGGCTAGGCAA SEQ ID NO. 19 — NS5B 8811-8830  C-10gTCCACAGTTACTCTCCAGG SEQ ID NO. 20 — NS5B 9019-9037  C-11gCCTCTTCAACTGGGCAGTA SEQ ID NO. 21 — NS5B 9170-9188  C-12AGCTTAAACTCACTCCAAT SEQ ID NO. 22 — NS5B 9196-9214 3′-1GCTCCATCTTAGCCCTAGT SEQ ID NO. 23 19 5-23* 3′-2 gTCCATCTTAGCCCTAGTCA SEQID NO. 24 19 7-25* 3′-3 GTCACGGCTAGCTGTGAAA SEQ ID NO. 25 19 22-40* 3′-4ACGGCTAGCTGTGAAAGGT SEQ ID NO. 26 19 25-43* 3′-5 GCTGTGAAAGGTCCGTGAG SEQID NO. 27 19 32-50* 3′-6 GGTCCGTGAGCCGCATGAC SEQ ID NO. 28 —41-59*{circumflex over ( )} 3′-7 GCCGCATGACTGCAGAGAGT SEQ ID NO. 29 —50-69*{circumflex over ( )} 3′-8 ACTGGCCTCTCTGCAGATCA SEQ ID NO. 30 —76-95*{circumflex over ( )} ^(‡)Lower case letters indicate sequencesnot corresponding to either the HCV fusion replicon or the HCV genome

TABLE 3 Luciferase-HCV fusion plasmids Luciferase-HCV Fusion Plasmid HCVTarget Region #20 5′1-through-5′5 #16 5′3-through-5′10 #175′6-through-5′10 #12 5′7-through-5′10, Coding-1 #18 Coding-3 #193′1-through-3′8 C2&4 Coding-2, Coding-4 C5 Coding-5 C6 Coding-6 C7Coding-7 C8 Coding-8 C9 Coding-9 C10 Coding-10 C11&12 Coding-11,Coding-12 C6-C9-C12-3′1 Coding-6, Coding-9, Coding-12, 3′1

To test the efficacy of the RNAi sequences selected, RNAi agents weredelivered directly to cultured cells along with a Luciferase-HCV fusionplasmid. A schematic representation of the Luciferase-HCV fusion plasmidused in these experiments is shown on the left panel of FIG. 9. Itcomprises a gene sequence coding for firefly luciferase protein fused to100 bp stretches of nucleic acid, corresponding to HCV targetsequences—the regions of HCV from which the RNAi agents were derived.RNAi agents directed against a sequence within the 100 bp region will,if effective, degrade the luciferase-HCV transcription product, thusdecreasing or eliminating luciferase expression. Table 3 lists some ofthe corresponding Luciferase-HCV fusion plasmids and the HCV targetregions used.

Pre-synthesized siRNA agents were obtained from Dharmacon, Inc.(Lafayette, Colo.). Huh7 cells were seeded in 12-well plates at 9.5×10⁵cells per well 24 hours before the time of transfection. At the time oftransfection the cells were ˜30-40% confluent. 350 μl OptiMEM(Invitrogen Inc.) media was mixed with 15 μl NovaFECTOR (VennNova,Pompano Beach, Fla.), and incubated for 1 hour at room temperature. 50μl OptiMEM was mixed with 0.05 μg pRL-SV40 (Promega), 0.45 μg of theLuc-HCV reporter plasmid, and 2 μl of the appropriate RNAi agent (20 μMstock). NovaFECTOR solution was added to the DNA/RNAi mixture, andincubated for 15 minutes at room temperature. Cells were rinsed oncewith OptiMEM and transfected with 400 μl of the NovaFECTOR/DNA/RNAimixture. Cells were then incubated at 37° C. in 5% CO₂ atmosphere for1.5 hours. One milliliter of complete medium was added to each well, andincubation was continued for an additional 2.5 hours, at which time themedium was replaced with a fresh complete medium. Further incubation wascontinued for two days.

After two days, the medium was aspirated and the cells were lysed andmeasured for luciferase expression according to the manufacturer's dualluciferase protocol (Promega, Madison, Wis.). Percent of inhibition wascalculated based on normalized luciferase relative light units (RLUs)versus cells transfected with a non-specific RNAi species with no knownendogenous target. The data was normalized for differences intransfection efficiency based on expression of Renilla luciferaseactivity from a pRL-SV40 plasmid that was co-transfected with theLuciferase-HCV fusion plasmids.

FIG. 12 shows the results of inhibition of luciferase expressionmeasured in relative light units by different RNAi agents targeting fivedifferent 100 bp regions of HCV; and FIG. 13 shows the results ofinhibition of luciferase expression where the data of FIG. 12 isexpressed as a percent value. In looking at these results, note that atleast 50% inhibition was achieved with all selected RNAi agents, andover half of the plasmids inhibited luciferase expression by greaterthan 80%.

FIG. 14 shows the reproducibility of the results of experimentsperformed testing four different RNAi agents (5′-1, 5′-2, 5′-3, and5′-4) targeting various segments of a 100 bp sequence in the 5′ regionof HCV. Note that reproducibility was excellent for each agent tested.

FIG. 15 shows the change in percent inhibition of luciferase expression24 and 48 hours post transfection for five different RNAi agents (5′-3,5′-4, 5′-5, 5′-6 and 5′-7) targeting various segments of a 100 bpsequence in the 5′ region of HCV.

FIG. 16 shows the change in percent inhibition of luciferase expression44 and 72 hours post transfection for two different RNAi agentstargeting various segments of a 100 bp sequence in the 5′ region of HCV(5′-1 and 5′-3), five different RNAi agents targeting various segmentsof a 100 bp sequence in the 3′ region of HCV (3′-1, 3′-2, 3′-3, 3′-4 and3′-5), and one RNAi agent targeting a segment of a 100 bp sequence inthe open reading frame region of HCV (C-3). Inhibition is maintained at72 hours post infection to within about 10 percent of 44 hour levels.

FIG. 18 shows the luciferase inhibition resulting from treatment with awide variety of RNAi agents targeted to various regions in the HCVgenome in a luciferase-HCV fusion plasmid. Luciferase activity ismeasured 48 hours after co-transfection with an RNAi agent into Huh7cells. It can be seen from this data that RNAi agents can effectivelytarget all regions of the HCV genome and result in strong inhibition ofluciferase reporter signal.

Example 3B Selection and Testing of RNAi Agents Against Luciferase-HCVReplicon System

Although many of the individual steps of HCV replication are understood,until recently there was no tissue culture system that propagated theviral life cycle, making studies of the virus difficult. However, an invitro replicon system has been developed (see, e.g., U.S. Pat. Nos.5,585,258; 6,472,180; and 6,127,116 to Rice, et al.). A replicon is anautonomously replicating portion of HCV genomic RNA that may contains amarker gene for selection and verification of replication. HCV-RNAconstructs are transfected into cell lines that are amenable to supportcontinuous propagation. Following the steps of the infectious cycle, theRNA is translated by the cellular machinery and produces both theappropriate viral proteins required for replication of the genome, aswell as the selectable marker, if present. Full-length and sub-genomicreplicons have been generated and shown to be functional, although onlythe non-structural proteins are obligate. The autonomously replicatingproperties of the RNA remain independent of expression of the structuralgenes. Even when present in replicons expressing the full length HCVgenome, the core and envelope proteins fail to effectively package thegenome into infectious particles—resulting in the loss of a model systemto study the packaging, egress and re-entry steps of the virus.Regardless, the replicon is able to recreate a portion of the biologyand mechanisms utilized by HCV.

In addition to or as an alternative to using luciferase or other suchreporters, the level of replicon activity may measured by a variety ofother methods. The inhibition of HCV replication may be assessed byobservation of the relative levels of non-structural proteins byimmunofluorescence microscopy utilizing a panel of commerciallyavailable HCV-specific monoclonal antibodies. Alternatively or inaddition, Q-PCR may be used to measure the relative level of HCV genomicRNA from each transfected condition.

The ability of siRNA agents to inhibit replication of the genomic RNAwas tested by measurement of renilla luciferase expression in a cellline transformed by the Luciferase-HCV fusion replicon. Five siRNAscould not be tested in the subgenomic replicon system due to an absenceof the corresponding sequence and were included in the tests merely asnonspecific controls. Schematic diagrams of the Luciferase-HCV repliconsare shown in FIGS. 8B and 8C. Cells were seeded in a 96-well plate;following 24 hour incubation, interferon alpha 2B (IFN), known toinhibit HCV replicon activity (Blight, et al. Science. 290: 1972-74(2000)), was added into specified wells at a concentration of 100 unitsper ml. Following an additional 48 hours of incubation, the medium wasdiscarded and a cell extract generated in situ and luciferase activitywas measured. Luciferase activity corresponded precisely to the levelsof luciferase-mRNA (data not shown). In testing RNAi agents, 29Σ cellscontaining the Luciferase-HCV replicon (shown in FIG. 8B) aretransfected with RNAi agents targeting a variety of regions in the HCVgenome. Cells are harvested after 48 hours, extracts generated and therelative level of luciferase activity assessed. FIG. 19 shows theresults of luciferase inhibition by siRNA agents directed to variousregions of the Luciferase-HCV replicon. RNAi agents directed at codingregions C-1 through C-5 do not have targets in the Luciferase-HCVreplicon and serve as additional non-specific controls. Once againtargets in all regions of the HCV genome can act as effective inhibitionsites for siRNA agents.

Once several highly functional RNAi are selected and testedindividually, they are then triple transfected into cells harboring thereplicon system. One control consists of transfecting an equivalentnumber of unrelated RNAi species in parallel. The inhibitory activity ofthe triple transfections is compared to activity from a set of parallelplates that have been transfected with only one RNAi species.

Three RNAi agents are validated, and the coding sequences for eachcorresponding shRNA is generated from long, complementary self-annealingoligonucleotides and cloned into the individual sites of the triplepromoter AAV vector. This construct is then packaged into viralparticles according to the methods described herein utilizing the systemthat results in the highest transduction efficiency of liver tissues.The total length of each promoter/RNAi/terminator component of thetriple promoter cassette is small (˜400 nucleotides); linking threepromoter/RNAi/terminator components together results in a sequence thatis 1200-1300 nucleotides in length, far below the upper size limit ofself-complementary AAV.

The inhibitory activity of these particles is tested on cell linesharboring the replicon. Generation of a triple promoter constructexpressing three unrelated shRNA species serves as a negative control.The efficacy of the shRNA sequences is monitored by aforementionedanalysis techniques.

Example 4 Development of a Triple Promoter Expression Construct

Construction of a triple promoter expression construct includes threeindependent promoter and terminator sequences that drive the expressionof the individual shRNA species at comparable levels of abundance.Repetition of promoter elements may leave integrated expressioncassettes susceptible to recombination events; thus, three distinctpromoters and terminators are identified and validated. The synthesis ofsmall nuclear RNAs and transfer RNAs is directed by RNA polymerase III(pol III) under the control of pol III-specific promoters. Because ofthe relatively high abundance of transcripts directed by theseregulatory elements, pol III promoters, including those derived from theU6 and H1 genes, have been used to drive the expression of shRNA (see,e.g., Domitrovich and Kunkel. Nucl. Acids Res. 31(9): 2344-52 (2003);Boden, et al. Nucl. Acids Res. 31(17): 5033-38 (2003a); and Kawasaki, etal. Nucleic Acids Res. 31(2): 700-7 (2003)).

Initially, the assessment of relative promoter strength of the polIII-specific sequences is conducted in vectors containing the single,individual promoters (shown in FIG. 7). Each promoter construct drivesexpression of a shRNA that has demonstrated functional inhibition ofluciferase activity (Elbashir, et al. Nature. 411: 494-498 (2001 a)).Since there is a wealth of data demonstrating the successful utilizationof the U6 promoter for the expression of shRNA, it is used as thestandard for assessing the relative strength of other promoters. Themajority of the promoters that are tested are quite short, most in therange of 200-300 nucleotides in length. Long, overlappingoligonucleotides are used to assemble the promoters and terminators denovo and are then cloned into multiple cloning sites that flank theshRNA. The promoter is paired with the termination signal that occursnaturally downstream of the gene from which the promoter is taken.

The relative strength of each promoter is assessed in vitro by thedecrease in activity of a co-transfected commercially availableluciferase reporter, pGL3Control (shown in FIG. 7) or pRLSV40 (Promega,Madison, Wis.). The test and reporter constructs are transfected intopermissive cells utilizing standard techniques. Controls consist of atest promoter construct in which the functional shRNA against luciferaseis replaced by an unrelated shRNA sequence. A third construct encodingfor the secreted protein human α1-antitrypsin (hAAT) is co-transfectedinto the cells in order to assess for variations in transfectionefficiencies. For assessing inhibitory activity of the shRNA, luciferaseactivity is measured utilizing standard commercial kits (Promega,Madison, Wis.). The shRNA-mediated decrease in luciferase expression,normalized to hAAT levels, is an indirect measurement of promoterstrength. Alternatively or in addition, quantitative real time PCRanalysis (Q-PCR) on luciferase RNA levels is performed on RNA that isharvested and purified from parallel experimental plates.

Once appropriate promoter and terminator pairs are identified, the finaltriple promoter RNAi expression cassette is designed. Several designs ofthe final vector are tested, including having all three promoters in atandem array or arranged in clockwise and counterclockwiseconfigurations (i.e., transcribed from the top and the bottom strand ofthe cassette DNA) or any variation thereof. Three such configurationsare shown in FIGS. 3A, 3B and 3C.

The configurations shown in FIGS. 3B and 3C were transfected into cellsand tested for inhibitory activity utilizing luciferase activity assays.Two or more promoters driving distinct RNAi species may result in anadditive or synergistic inhibitory effect, thus, in order to assess thefunctionality and relative strength of each of the promoters within thecontext of the triple promoter expression construct, variants of theexpression cassettes was generated as detailed in Table 4. Utilizingthese species, the inhibitory effect of the shRNA driven from eachpromoter within the triple expression construct was measured byluciferase assays. Alternatively, Q-PCR is used to assess relativelevels of transcript driven by each promoter. Although theself-complementary nature of hairpin-RNA generally would prevent thedirect Q-PCR measurement of these RNA transcripts, three differentnon-hairpin transcripts of approximately the same size can besubstituted into the vectors in place of the shRNA using viralmultiple-promoter RNAi expression constructs with cassettes such asthose shown in FIGS. 3D and 3E may be used.

The following RNA Pol III Class 3 promoters: U6-1, U6-8, U6-9, H1 Long,Human Y4, Human Y5, were selected, synthesized and cloned into either asingle or multiple promoter construct. The luciferase specific shRNA wasthen cloned downstream of the single promoter construct or downstream ofone of the promoters in the triple promoter construct (Table 4).

72 hours after transfection, the media was aspirated and the cells werelysed and measured for luciferase expression according to themanufacturer's dual luciferase protocol (Promega, Madison, Wis.).Percent of inhibition was calculated based on normalized luciferaserelative light units (RLUs) versus mock-treated cells (the negativecontrol). An unrelated RNAi agent was used as the mock/negative controlin each experiment. Normalization of luciferase RLUs for transfectionefficiency was made based on expression of Renilla luciferase expressedfrom pRL-SV40 plasmid co-transfected with the target plasmids.

All promoters were shown to be similarly active in both constructs ofFIG. 3C type in Huh 7 cells (17A), and constructs of FIG. 3B type in 293cells (17B). It can be seen that the inhibitory properties are similarfor shRNA in single and multiple promoter contexts. The data showed thatinhibition of the shRNA is comparable in a triple promoter context andin both Huh7 and 293 cells.

TABLE 4 Promoter/shRNA inserts in constructs of the type shown in FIG.3B and 3C used for assessing relative inhibitory contribution of eachexpression cassette Promoter/shRNA A B C Construct U6-1 U6-8 U6-9Construct I shRNA-LUC empty empty Construct II empty shRNA-LUC emptyConstruct III empty empty shRNA-LUC

Plasmids comprising the promoter/shRNA/terminator cassettes of the typeshown in FIG. 3B were used to assess the relative inhibitorycontribution of shRNA agents expressed from multiple promoter expressioncassettes. Plasmids comprising shRNA constructs targeting differentregions of the HCV genome are placed operably under the control ofdifferent promoters. Table 5 shows which shRNA are under the control ofthe separate promoters including shRNA agents under control of thepromoter at position B that was inactive. Luciferase-HCV fusion plasmid# 16 containing the HCV sequence from regions 5′-3 through 5′-10 linkedto the luciferase gene was co-transfected into Huh7 cells along withmultiple promoter constructs. FIG. 20 shows the increased inhibition ofluciferase when co-transfected with a multiple promoter/double shRNAexpression construct as compared to a construct comprising a singleshRNA linked to a single promoter.

TABLE 5 Promoter/shRNA inserts in constructs of the type shown in FIG.3B used for assessing relative inhibitory contribution of an individualand dual promoters in a multiple promoter expression cassette.Promoter/shRNA A B C Construct U6-9 inactive U6-8 Construct I 5-′3 5′-1empty Construct II 5-′3 C-12 empty Construct III 5-′3 5′-8 emptyConstruct IV 5-′3 5′-8 5′-6

Example 4B Testing of shRNA Triple Promoter Constructs in vitro

Triple promoter cassettes of the type shown in FIG. 3C were generatedwith the following promoters; U6-9 in position A, U6-1 in position B,and U6-8 in position C. The promoters drove transcription of shRNAsequences targeting various positions in the HCV genome or were followedby stretches of T's in the ‘empty’ configuration to prevent promoterread-through. Single promoter/shRNA constructs used as controls wereconstructed using the U6-1 promoter. The single or triple promoterconstructs were co-transfected with luciferase-HCV reporter plasmidsthat contained different HCV target regions. FIG. 21 shows the resultsof luciferase inhibition after co-transfection with single or multiplepromoter constructs that targeted different regions in the HCV genomeand one of three luciferase-HCV fusion plasmids containing sequence fromtarget regions in the HCV genome. Luciferase activity was measured 72hours after co-transfection into Huh7 cells. It can be seen in the topgraph in FIG. 21 that shRNA specific for the C-12 region of HCV showsthe appropriate inhibitory activity to luciferase reporter plasmidcontaining the coding region C-12 of HCV. No non-specific inhibition isobserved when shRNA specific for other regions of HCV are expressedeither singly or as part of a multiple promoter cassette of thisinvention. Similar results can be seen in the middle graph where theLuciferase-HCV reporter plasmid contains sequence from the coding regionC-9 of HCV. In this case, shRNA specific to the C-9 region expressedeither from a single promoter or triple promoter cassette had thestrongest inhibitory activity of the shRNA agents tested. The bottomgraph shows the luciferase inhibition resulting from co-transfectionwith single or triple promoter constructs and reporter plasmidscontaining the 5′6 region of HCV. It can be seen that the strongestinhibition results from constructs containing shRNA specific for the 5′6target in either a single or multiple promoter construct. Triplepromoter constructs of this invention work to effectively suppressspecific gene targets.

Example 5 Testing of shRNA Triple Promoter Constructs Agents in vivo

In vivo evaluation of multiple promoter constructs of this inventionwere assessed by co-transfection of mouse liver with the multiplepromoter/shRNA plasmid DNA of the type shown in FIG. 3C and theappropriate firefly luciferase-HCV fusion reporter plasmid using thehydrodynamic tail vein injection procedure. The multiple promoter/shRNAplasmid used here controlled the expression of shRNA species thattargeted the coding 9, coding 12 and the 5′8 position of HCV. Negativecontrol mice were injected with the reporter construct and an irrelevantshRNA. In addition, mice were injected with plasmids expressing therenilla luciferase protein. This protein was used to normalize thetransfection efficiency of mouse liver. Forty eight hours afterinjection, the animals were sacrificed, and the livers were harvested.Liver lysates were assayed for firefly luciferase activity and renillaluciferase activity using a Promega luciferase kit. Levels of inhibitioninduced by the shRNA expression from the hairpin constructs are assessedrelative to the negative control. The results shown in Table 6 show thattriple promoter constructs of this invention effectively inhibitreporter signal in vivo.

TABLE 6 Percent inhibition of firefly luciferase signal by triplepromoter/shRNA plasmid Reporter % Inhibition by Triple plasmid FireflyLuciferase shRNA Compared to Plasmid expressing (12 ug/ RLU (normalizednon-specific shRNA Group # n shRNA(5 ug/mouse) mouse) to Renilla)Control 21 5 Triple promoter/shRNA C-9 0.05 98% (5′8as, C-9s, C-12as)(pBen71) 23 5 Single non-specific 5′-3 C-9 3.20 n/a (non-specific shRNA)(pBen71) 25 5 Triple promoter/shRNA C-11/12 1.00 93% (5′8as, C-9s,C-12as) (pBen73) 27 5 Single non-specific 5′-3 C-11/12 15.10 n/a(non-specific shRNA) (pBen73)

Infectious AAV particles containing vectors that express the shRNAtargeted against HCV sequences are delivered to normal mice either bytail vein or hepatic portal vein injection. Infectious AAV particlesexpressing three unrelated shRNAs serve as the negative control.Initially, a fairly high dose of virus, e.g. 2×10¹² vector genomes, isused, though subsequent experiments are performed to establishdose-response curves. An appropriate firefly luciferase-HCV fusionreporter plasmid is injected at various times using the hydrodynamictail vein injection procedure. 48-72 hours after injection of thereporter plasmid, the mice are sacrificed, the livers harvested andsamples of serum collected. Firefly luciferase activity is used as abenchmark to assess efficacy of the AAV delivered shRNA. In addition,monitoring the serum levels of hAAT or liver levels of renillaluciferase are used to determine transfection efficiency betweenanimals. Serum levels of the liver enzymes alanine aminotransferase,aspartate aminotransferase, and tumor necrosis factor alpha are measuredto ensure general hepatic toxicity is not induced by the treatment.

Example 6 Testing AAV-delivery of shRNA Against an in vivo ReplicatingHBV Model System

There is no ideal small animal model for testing the efficacy ofAAV-delivered shRNA expression constructs against HCV. However, testingthe AAV expression RNAi construct in an alternate model system may beused to assess the extent of inhibition of viral replication in theliver. Although the sequence composition of the delivered shRNA isnecessarily different in such a model, the remainder of the system,including the AAV triple promoter expression vector and packagingcomponents, remain unchanged. The model system for the hepatitis B virus(HBV) is utilized. Selection of shRNA sequences to be included in thetriple promoter AAV expression construct to target HBV are chosen basedon the efficacy of shRNA sequences published to date (McCaffrey, et al.Nature Biotech. 21(6): 639-644 (2003); Ying, et al. Biochem. Biophys.Res. Commun. 309(2): 482-484 (2003); Klein, et al. Gastroent. 125(1):9-18 (2003); and Shlomai, et al. Hepatology. 37(4): 764-70 (2003)). Theappropriate AAV expression construct with the triple promoter drivingthree HBV-specific shRNAs is then packaged into viral particlesaccording to the methods described herein.

The mice are first injected by hydrodynamic transfection procedures withthe expression plasmids bearing the sequences of the HBV genome and areallowed to establish HBV replication. In order to assess transfectionefficiency, a plasmid encoding for hAAT is added into the mixture forco-injection. Initial indications of HBV activity is assessed by theappearance of Hepatitis B Virus surface antigen (HBsAg) and the HBV coreantigen (HBcAg) in the serum of the treated animals by ELISA assayscollected by retro-orbital plexus bleed.

Following establishment of HBV replication in the mouse livers, the AAVviral particles packaging the triple promoter constructs encoding HBVshRNAs are introduced into the mouse using tail vein injection orhepatic portal vein injection. AAV viral particles bearing an AAVexpression construct encoding three HBV-unrelated shRNAs are utilized asa negative control. At the termination of the experiment, serum samplesare monitored for down regulation of the quantities of HBsAg and HBcAgproteins. Alternatively or in addition, liver tissues are assessed forthe relative levels of HBV RNA by Q-PCR. Systemic liver toxicity isevaluated by monitoring serum for alanine aminotransferase, aspartateaminotransferase, and tumor necrosis factor alpha by ELISA.

FIG. 10 is a graphic illustration of one embodiment of a precursortriple promoter cassette showing unique restriction sites useful forinserting or removing RNAi species or terminator elements or forswitching out the U6-9, pU6 or U6-8 promoters shown. In addition, arrowsshow the direction of transcription of each of the threepromoter/RNAi/terminator components. FIGS. 11A and 11B/11C show thenucleotide sequence (SEQ ID NO 31 and 32, respectively) of twoembodiments of the precursor promoter cassettes of this invention. FIG.10, shows the locations of the U6-9, pU6 or U6-8 promoters, as well asthe restriction sites of a precursor promoter cassette of thisinvention.

While the present invention has been described with reference tospecific embodiments, it should be understood by those skilled in theart that various changes may be made and equivalents may be substitutedwithout departing from the true spirit and scope of the invention. Inaddition, many modifications may be made to adapt a particularsituation, material or process to the objective, spirit and scope of thepresent invention. All such modifications are intended to be within thescope of the invention.

All references cited herein are to aid in the understanding of theinvention, and are incorporated in their entireties for all purposeswithout limitation.

1. A method of inhibiting the level of one or more Hepatitis C virusnucleic acid targets that are expressed in a cell comprising contactingthe cell with a genetic construct comprising a multi-promoter expressioncassette, the multi-promoter expression construct comprising at leastthree promoter/RNAi/terminator components wherein eachpromoter/RNAi/terminator component comprises a promoter element, aterminator element and an RNAi sequence, the RNAi sequence operablylinked to the promoter element and terminator element, and wherein theRNAi sequences are SEQ ID NO: 6, 19 and
 22. 2. A method of treating ananimal cell, tissue or organ, to inhibit the level of one or moreHepatitis C virus nucleic acid targets expressed in a cell, said methodcomprising introducing to said animal cell, tissue or organ a geneticconstruct comprising a multi-promoter expression cassette, themulti-promoter expression cassette comprising at least threepromoter/RNAi/terminator components wherein eachpromoter/RNAi/terminator component comprises a promoter element, aterminator element and an RNAi sequence operably linked to the promoterelement and the terminator element, wherein the RNAi sequences are SEQID NO:6, 19 and 22.